Compositions and Methods of Modulating Short-Chain Dehydrogenase Activity

ABSTRACT

Compounds and methods of modulating 15-PGDH activity, modulating tissue prostaglandin levels, treating disease, diseases disorders, or conditions in which it is desired to modulate 15-PGDH activity and/or prostaglandin levels include 15-PGDH inhibitors described herein.

RELATED APPLICATION

This application claims priority from U.S. Provisional Application Nos.61/891,260, filed Oct. 15, 2013, 61/954,202, filed Mar. 17, 2014,62/019,597, filed Jul. 1, 2014, and 62/043,694, filed Aug. 29, 2014, thesubject matter of which are incorporated herein by reference in theirentirety.

GOVERNMENT FUNDING

This invention was made with government support under Grant Nos.R01CA127306, R01CA127306-03S1, 1P01CA95471-10, AND 5P50CA150964, awardedby The National Institutes of Health. The United States government mayhave certain rights to the invention.

BACKGROUND

Short-chain dehydrogenases (SCDs) are a family of dehydrogenases thatshare only 15% to 30% sequence identity, with similarity predominantlyin the coenzyme binding domain and the substrate binding domain. Inaddition to their role in detoxification of ethanol, SCDs are involvedin synthesis and degradation of fatty acids, steroids, and someprostaglandins, and are therefore implicated in a variety of disorderssuch as lipid storage disease, myopathy, SCD deficiency, and certaingenetic disorders.

The SCD, 15-hydroxy-prostaglandin dehydrogenase (15-PGDH),(hydroxyprostaglandin dehydrogenase 15-(nicotinamideadeninedinucleotide); 15-PGDH; Enzyme Commission number 1.1.1.141;encoded by the HPGD gene), represents the key enzyme in the inactivationof a number of active prostaglandins, leukotrienes andhydroxyeicosatetraenoic acids (HETEs) (e.g., by catalyzing oxidation ofPGE₂ to 15-keto-prostaglandin E2, 15k-PGE). The human enzyme is encodedby the HPGD gene and consists of a homodimer with subunits of a size of29 kDa. The enzyme belongs to the evolutionarily conserved superfamilyof short-chain dehydrogenase/reductase enzymes (SDRs), and according tothe recently approved nomenclature for human enzymes, it is namedSDR36C1. Thus far, two forms of 15-PGDH enzyme activity have beenidentified, NAD+-dependent type I 15-PGDH that is encoded by the HPGDgene, and the type II NADP-dependent 15-PGDH, also known as carbonylreductase 1 (CBR1, SDR21C1). However, the preference of CBR1 for NADPand the high Km values of CBR1 for most prostaglandin suggest that themajority of the in vivo activity can be attributed to type I 15-PGDHencoded by the HPGD gene, that hereafter, and throughout all followingtext, simply denoted as 15-PGDH.

Recent studies suggest that inhibitors of 15-PGDH and activators of15-PGDH could be therapeutically valuable. It has been shown that thereis an increase in the incidence of colon tumors in 15-PGDH knockoutmouse models. A more recent study implicates increased 15-PGDHexpression in the protection of thrombin-mediated cell death. It is wellknown that 15-PGDH is responsible for the inactivation of prostaglandinE2 (PGE₂), which is a downstream product of COX-2 metabolism. PGE₂ hasbeen found to be neurotoxic both in vitro and in vivo; thus, COX-2specific inhibitors, which decrease PGE₂ release, exhibitneuroprotective effects. PGE₂ has also been shown to be beneficial in avariety of biological processes, such as hair density, dermal woundhealing, and bone formation.

SUMMARY

Embodiments described herein relate to compounds and methods ofmodulating short chain dehydrogenase (SCD) (e.g., 15-PGDH) activities,modulating tissue prostaglandin levels, and/or treating diseases,disorders, or conditions in which it is desired to modulate SCD (e.g.,15-PGDH) activity and/or prostaglandin levels.

In some embodiments, the modulator of SCD can be an SCD inhibitor thatcan be administered to tissue or blood of a subject at an amounteffective to inhibit the activity of a short chain dehydrogenase enzyme.The SCD inhibitor can be a 15-PGDH inhibitor that can be administered totissue or blood of a subject at an amount effective to increaseprostaglandin levels in the tissue or blood. The 15-PGDH inhibitor caninclude a compound having the formula (V):

-   -   wherein n=0-2;    -   R₁ and R₃ are the same or different and are each selected from        the group consisting of:

-   -   R₂ is N or CR₇;    -   R₄ is selected from the group consisting of H, Cl, F, NH₂, and        N(R₆)₂;    -   R₅ is selected from the group consisting of branched or linear        alkyl including —(CH₂)n₁CH₃ (n₁=0-7),

wherein n₂=0-6 and X is any of the following: CF_(y)H_(z) (y+z=3),CCl_(y)H_(z) (y+z=3), OH, OAc, OMe, R₆, OR₆, CN, N(R₆)₂,

(n₃=0-5, m=1-5), and

(n₄=0-5);

-   -   each R₆ and R₇ are the same or different and are one or more        substituent selected from the group consisting of hydrogen,        substituted or unsubstituted C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl,        C₂-C₂₄ alkynyl, C₃-C₂₀ aryl, heterocycloalkenyl containing from        5-6 ring atoms, (wherein from 1-3 of the ring atoms is        independently selected from N, NH, N(C₁-C₆ alkyl), NC(O)(C₁-C₆        alkyl), O, and S), heteroaryl or heterocyclyl containing from        5-14 ring atoms, (wherein from 1-6 of the ring atoms is        independently selected from N, NH, N(C₁-C₃ alkyl), O, and S),        C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, halo, silyl, hydroxyl,        sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy,        C₅-C₂₀ aryloxy, acyl (including C₂-C₂₄ alkylcarbonyl (—CO-alkyl)        and C₆-C₂₀ arylcarbonyl (—CO-aryl)), acyloxy (—O-acyl), C₂-C₂₄        alkoxycarbonyl (—(CO)—O-alkyl), C₆-C₂₀ aryloxycarbonyl        (—(CO)—O-aryl), C₂-C₂₄ alkylcarbonato (—O—(CO)—O-alkyl), C₆-C₂₀        arylcarbonato (—O—(CO)—O-aryl), carboxy (—COOH), carboxylato        (—COO⁻), carbamoyl (—(CO)—NH₂), C₁-C₂₄ alkyl-carbamoyl        (—(CO)—NH(C₁-C₂₄ alkyl)), arylcarbamoyl (—(CO)—NH-aryl),        thiocarbamoyl (—(CS)—NH₂), carbamido (—NH—(CO)—NH₂), cyano        (—CN), isocyano (—N⁺C⁻), cyanato (—O—CN), isocyanato (—O—N⁺═C⁻),        isothiocyanato (—S—CN), azido (—N═N⁺═N⁻), formyl (—(CO)—H),        thioformyl (—(CS)—H), amino (—NH₂), C₁-C₂₄ alkyl amino, C₅-C₂₀        aryl amino, C₂-C₂₄ alkylamido (—NH—(CO)-alkyl), C₆-C₂₀ arylamido        (—NH—(CO)-aryl), sulfanamido (—SO2NR2 where R is independently        H, alkyl, aryl or heteroaryl), imino (—CR═NH where R is        hydrogen, C₁-C₂₄ alkyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄        aralkyl, etc.), alkylimino (—CR═N(alkyl), where R=hydrogen,        alkyl, aryl, alkaryl, aralkyl, etc.), arylimino (—CR═N(aryl),        where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (—NO₂),        nitroso (—NO), sulfo (—SO₂—OH), sulfonato (—SO₂—O⁻), C₁-C₂₄        alkylsulfanyl (—S-alkyl; also termed “alkylthio”), arylsulfanyl        (—S-aryl; also termed “arylthio”), C₁-C₂₄ alkylsulfinyl        (—(SO)-alkyl), C₅-C₂₀ arylsulfinyl (—(SO)-aryl), C₁-C₂₄        alkylsulfonyl (—SO₂-alkyl), C₅-C₂₀ arylsulfonyl (—SO₂-aryl),        sulfonamide (—SO₂—NH2, —SO₂NY₂ (wherein Y is independently H,        aryl or alkyl), phosphono (—P(O)(OH)₂), phosphonato        (—P(O)(O⁻)₂), phosphinato (—P(O)(O⁻)), phospho (—PO₂), phosphino        (—PH₂), polyalkyl ethers (—[(CH₂)_(n)O]_(m)), phosphates,        phosphate esters [—OP(O)(OR)₂ where R=H, methyl or other alkyl],        groups incorporating amino acids or other moieties expected to        bear positive or negative charge at physiological pH, and        combinations thereof;    -   R₃ is not hydrogen if R₁ is H, an unsubstituted thiophene, or an        unsubstituted thiazole and R₅ is butyl; or R₃ is not an        unsubstituted phenyl if R₁ is H, or an unsubstituted phenyl,        thiophene, or thiazole and R₅ is benzyl or (CH₂)n₅(CH₃)        (n₅=0-5); and pharmaceutically acceptable salts thereof.

In some embodiments, R₂ can be N or CH. R₁ can be a substituted orunsubstituted heterocyclyl containing 5-6 ring atoms. For example, R₁can be a substituted or unsubstituted thiophene, thiazole, oxazole,imidazole, pyridine, or phenyl. R₃ can be selected from the groupconsisting of H, substituted or unsubstituted aryl, a substituted orunsubstituted cycloalkyl, and a substituted or unsubstitutedheterocyclyl, alkyl, or carboxy including carboxylic acid (—CO2H),carboxy ester (—CO₂alkyl) and carboxamide [—CON(H)(alkyl) or—CO₂N(alkyl)₂].

In still other embodiments, the 15-PGDH inhibitor can include a compoundhaving formula (V₁):

-   -   wherein n=0-2;    -   R₃ is selected from the group consisting of:

-   -   R₂ is N or CR₇;    -   R₄ is selected from the group consisting of H, Cl, F, NH₂, and        N(R₆)₂;    -   R₅ is selected from the group consisting of branched or linear        alkyl including —(CH₂)n₁CH₃ (n₁=0-7),

wherein n₂=0-6 and X is any of the following: CF_(y)H_(z) (y+z=3),CCl_(y)H_(z) (y+z=3), OH, OAc, OMe, R₆, OR₆, CN, N(R₆)₂,

(n₃=0-5, m=1-5), and

(n₄=0-5);

-   -   each R₆ and R₇ are the same or different and are one or more        substituent selected from the group consisting of hydrogen,        substituted or unsubstituted C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl,        C₂-C₂₄ alkynyl, C₃-C₂₀ aryl, heterocycloalkenyl containing from        5-6 ring atoms, (wherein from 1-3 of the ring atoms is        independently selected from N, NH, N(C₁-C₆ alkyl), NC(O)(C₁-C₆        alkyl), O, and S), heteroaryl containing from 5-14 ring atoms,        (wherein from 1-6 of the ring atoms is independently selected        from N, NH, N(C₁-C₃ alkyl), O, and S), C₆-C₂₄ alkaryl, C₆-C₂₄        aralkyl, halo, silyl, hydroxyl, sulfhydryl, C₁-C₂₄ alkoxy,        C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀ aryloxy, acyl        (including C₂-C₂₄ alkylcarbonyl (—CO-alkyl) and C₆-C₂₀        arylcarbonyl (—CO-aryl)), acyloxy (—O-acyl), C₂-C₂₄        alkoxycarbonyl (—(CO)—O-alkyl), C₆-C₂₀ aryloxycarbonyl        (—(CO)—O-aryl), C₂-C₂₄ alkylcarbonato (—O—(CO)—O-alkyl), C₆-C₂₀        arylcarbonato (—O—(CO)—O-aryl), carboxy (—COOH), carboxylato        (—COO⁻), carbamoyl (—(CO)—NH₂), C₁-C₂₄ alkyl-carbamoyl        (—(CO)—NH(C₁-C₂₄ alkyl)), arylcarbamoyl (—(CO)—NH-aryl),        thiocarbamoyl (—(CS)—NH₂), carbamido (—NH—(CO)—NH₂), cyano        (—CN), isocyano (—N⁺C⁻), cyanato (—O—CN), isocyanato (—O—N⁺═C⁻),        isothiocyanato (—S—CN), azido (—N═N⁺═N⁻), formyl (—(CO)—H),        thioformyl (—(CS)—H), amino (—NH₂), C₁-C₂₄ alkyl amino, C₅-C₂₀        aryl amino, C₂-C₂₄ alkylamido (—NH—(CO)-alkyl), C₆-C₂₀ arylamido        (—NH—(CO)-aryl), sulfanamido (—SO2NR2 where R is independently        H, alkyl, aryl or heteroaryl), imino (—CR═NH where R is        hydrogen, C₁-C₂₄ alkyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄        aralkyl, etc.), alkylimino (—CR═N(alkyl), where R=hydrogen,        alkyl, aryl, alkaryl, aralkyl, etc.), arylimino (—CR═N(aryl),        where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (—NO₂),        nitroso (—NO), sulfo (—SO₂—OH), sulfonato (—SO₂—O⁻), C₁-C₂₄        alkylsulfanyl (—S-alkyl; also termed “alkylthio”), arylsulfanyl        (—S-aryl; also termed “arylthio”), C₁-C₂₄ alkylsulfinyl        (—(SO)-alkyl), C₅-C₂₀ arylsulfinyl (—(SO)-aryl), C₁-C₂₄        alkylsulfonyl (—SO₂-alkyl), C₅-C₂₀ arylsulfonyl (—SO₂-aryl),        sulfonamide (—SO₂—NH2, —SO₂NY₂ (wherein Y is independently H,        aryl or alkyl), phosphono (—P(O)(OH)₂), phosphonato        (—P(O)(O⁻)₂), phosphinato (—P(O)(O⁻)), phospho (—PO₂), phosphino        (—PH₂), polyalkyl ethers (—[(CH₂)_(n)O]_(m)), phosphates,        phosphate esters [—OP(O)(OR)₂ where R=H, methyl or other alkyl],        groups incorporating amino acids or other moieties expected to        bear positive or negative charge at physiological pH, and        combinations thereof;    -   R₃ is not hydrogen if R₅ is butyl; or R₃ is not an unsubstituted        phenyl if R₅ is benzyl or (CH₂)n₅(CH₃) (n₅=0-5); and        pharmaceutically acceptable salts thereof.

In some embodiments, the 15-PGDH inhibitor can inhibit the enzymaticactivity of recombinant 15-PGDH at an IC₅₀ of less than 1 μM, orpreferably at an IC₅₀ of less than 250 nM, or more preferably at an IC₅₀of less than 50 nM, or more preferably at an IC₅₀ of less than 10 nM, ormore preferably at an IC₅₀ of less than 5 nM at a recombinant 15-PGDHconcentration of about 5 nM to about 10 nM.

The 15-PGDH inhibitor can be provided in a topical composition that canbe applied to skin of a subject to promote and/or stimulate pigmentationof the skin and/or hair growth and/or inhibiting hair loss, and/or treatskin damage or inflammation.

The 15-PGDH inhibitor can also be administered to a subject to promotewound healing, tissue repair, and/or tissue regeneration and/orengraftment or regeneration of a tissue graft.

In one embodiment, the 15-PGDH inhibitor can be administered to asubject to treat at least one of oral ulcers, gum disease, colitis,ulcerative colitis, gastrointestinal ulcers, inflammatory bowel disease,vascular insufficiency, Raynaud's disease, Buerger's disease, diabeticneuropathy, pulmonary artery hypertension, cardiovascular disease, andrenal disease.

In another embodiment, the 15-PGDH inhibitor can be administered to asubject in combination with a prostanoid agonist for the purpose ofenhancing the therapeutic effect of the agonist in prostaglandinresponsive conditions.

In other embodiments, the 15-PGDH inhibitor can be administered to asubject and/or tissue of the subject to increase tissue stem cells. Forexample, the 15-PGDH inhibitor can be administered to bone marrow of asubject to increase stem cells in the subject.

In still other embodiments, the 15-PGDH inhibitor can be administered toa tissue graft donor, bone marrow graft donor, and/or a hematopoieticstem cell donor, and/or a tissue graft, and/or a bone marrow graft,and/or a hematopoietic stem cell graft, to increase the fitness of adonor tissue graft, a donor bone marrow graft, and/or a donorhematopoietic stem cell graft. For example, the 15-PGDH inhibitor can beadministered to a subject, and/or bone marrow of a subject to increasethe fitness of the marrow as a donor graft, and/or to a preparation ofhematopoietic stem cells of a subject to increase the fitness of thestem cell preparation as a donor graft, and/or to a preparation ofperipheral blood hematopoietic stem cells of a subject to increase thefitness of the stem cell preparation as a donor graft, and/or to apreparation of umbilical cord blood stem cells to increase the fitnessof the stem cell preparation as a donor graft, and/or to a preparationof umbilical cord blood stem cells to decrease the number of units ofumbilical cord blood required for transplantation.

In other embodiments, the 15-PGDH inhibitor can be administered to asubject to mitigate tissue graft rejection, to enhance tissue and/orbone marrow graft engraftment, to enhance bone marrow graft engraftment,following treatment of the subject or the marrow of the subject withradiation therapy, chemotherapy, or immunosuppressive therapy, toenhance engraftment of a progenitor stem cell graft, hematopoietic stemcell graft, or an umbilical cord blood stem cell graft, to enhanceengraftment of a hematopoietic stem cell graft, or an umbilical cordstem cell graft, following treatment of the subject or the marrow of thesubject with radiation therapy, chemotherapy, or immunosuppressivetherapy, and/or in order to decrease the number of units of umbilicalcord blood required for transplantation into the subject.

In other embodiments, the 15-PGDH inhibitor can be administered to arecipient of a tissue graft transplant, bone marrow transplant, and/orhematopoietic stem cell transplant, or of an umbilical cord stem celltransplant, in order to decrease the administration of other treatmentsor growth factors.

In some embodiments, the 15-PGDH inhibitor can be administered to asubject or to a tissue graft of a subject to mitigate graft rejection,to enhance graft engraftment, and/or to enhance graft engraftmentfollowing treatment of the subject or the marrow of the subject withradiation therapy, chemotherapy, or immunosuppressive therapy.

In other embodiments, the 15-PGDH inhibitor can be administered to asubject or to the bone marrow of a subject to confer resistance to toxicor lethal effects of exposure to radiation, to confer resistance to thetoxic effect of Cytoxan, the toxic effect of fludarabine, the toxiceffect of chemotherapy, or the toxic effect of immunosuppressivetherapy, to decrease pulmonary toxicity from radiation, and/or todecrease infection.

In still other embodiments, the 15-PGDH inhibitor can be administered toa subject to increase neutrophil counts following a hematopoetic celltransplant with bone marrow, hematopoetic stem cells, or umbilical cordblood, to increase neutrophil counts in a subject with neutropiafollowing chemotherapy administration or radiation therapy, to increaseneutrophil counts in a subject with aplastic anemia, myelodysplasia,myelofibrosis, neutropenia due to other bone marrow diseases, druginduced neutropenia, autoimmune neutropenia, idiopathic neutropenia, orneutropenia following viral infections, to increase neutrophil counts ina subject with neutropia, to increase platelet counts following ahematopoetic cell transplant with bone marrow, hematopoetic stem cells,or umbilical cord blood, to increase platelet counts in a subject withthrombocytopenia following chemotherapy administration or radiationtherapy, to increase platelet counts in a subject with aplastic anemia,myelodysplasia, myelofibrosis, thrombocytopenia due to other bone marrowdiseases, drug induced thrombocytopenia, autoimmune thrombocytopenia,idiopathic thrombocytopenic purpura, idiopathic thrombocytopenia, orthrombocytopenia following viral infections, to increase platelet countsin a subject with thrombocytopenia, to increase red blood cell counts,or hematocrit, or hemoglobin level, following a hematopoetic celltransplant with bone marrow, hematopoetic stem cells, or umbilical cordblood, to increase red blood cell counts, or hematocrit, or hemoglobinlevel in a subject with anemia following chemotherapy administration orradiation therapy, to increase red blood cell counts, or hematocrit, orhemoglobin level counts in a subject with aplastic anemia,myelodysplasia, myelofibrosis, anemia due to other disorder of bonemarrow, drug induced anemia, immune mediated anemias, anemia of chronicdisease, anemia following viral infections, or anemia of unknown cause,to increase red blood cell counts, or hematocrit, or hemoglobin level ina subject with anemia, to increase bone marrow stem cells, following ahematopoetic cell transplant with bone marrow, hematopoetic stem cells,or umbilical cord blood, to increase bone marrow stem cells in a subjectfollowing chemotherapy administration or radiation therapy, and/or toincrease bone marrow stem cells in a subject with aplastic anemia,myelodysplasia, myelofibrosis, other disorder of bone marrow, druginduced cytopenias, immune cytopenias, cytopenias following viralinfections, or cytopenias.

In other embodiments, the 15-PGDH inhibitor can be administered to asubject to increase responsiveness to cytokines in the presence ofcytopenias, with cytopenias including any of: neutropenia,thrombocytopenia, lymphocytopenia and anemia; and with cytokines havingincreased responsiveness potentiated by the 15-PGDH inhibitor includingany of: G-CSF, GM-CSF, EPO, IL-3, IL-6, TPO, TPO-RA (thrombopoietinreceptor agonist), and SCF.

In some embodiments, the 15-PGDH inhibitor can be administered to asubject to increase bone density, treat osteoporosis, promote healing offractures, or promote healing after bone surgery or joint replacementand/or to promote healing of bone to bone implants, bone to artificialimplants, dental implants, and bone grafts.

In other embodiments, the 15-PGDH inhibitor can be administered to asubject or to the intestine of a subject to increase stem cells or cellproliferation in the intestine and/or and confer resistance to toxic orlethal effects of exposure to radiation or the toxic, lethal, ormucositis effects resultant from treatment with chemotherapy.

In some embodiments, the 15-PGDH inhibitor can be administered to asubject or to intestine of a subject as a treatment for colitis,ulcerative colitis, or inflammatory bowel disease.

In other embodiments, the 15-PGDH inhibitor can be administered to asubject to increase liver regeneration following liver surgery,following live liver donation, following liver transplantation, orfollowing liver injury by toxins and/or to promote recovery from orresistance to liver toxins, including acetaminophen and relatedcompounds.

In still other embodiments, the 15-PGDH inhibitor can be administered toa subject to treat erectile dysfunction.

In yet other embodiments, the 15-PGDH inhibitor can be administered toinhibit at least one of the growth, proliferation, or metastasis of15-PGDH expressing cancers.

Still other embodiments described herein relate to a method of treatinga subject in need of cell therapy. The method includes administering tothe subject a therapeutically effective amount of a preparationcomprising human hematopoietic stem cell administered a 15-PGDHinhibitor described herein and/or a therapeutic composition comprisinghuman hematopoietic stem cells and a 15-PGDH inhibitor described herein.

In some embodiments, the subject has received human hematopoietic stemcells and/or has received the preparation and/or the therapeuticcomposition.

In other embodiments, the subject has acute myelogenous leukemia (AML),acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML),chronic lymphocytic leukemia (CLL), juvenile myelomonocytic leukemia,Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, severeaplastic anemia, Fanconi's anemia, paroxysmal nocturnal hemoglobinuria(PNH), pure red cell aplasia, amegakaryocytosis/congenitalthrombocytopenia, severe combined immunodeficiency syndrome (SCID),Wiskott-Aldrich syndrome, beta-thalassemia major, sickle cell disease,Hurler's syndrome, adrenoleukodystrophy, metachromatic leukodystrophy,myelodysplasia, refractory anemia, chronic myelomonocytic leukemia,agnogenic myeloid metaplasia, familial erythrophagocyticlymphohistiocytosis, solid tumors, chronic granulomatous disease,mucopolysaccharidoses, or Diamond Blackfan anemia.

Other embodiments relate to a method of treating a subject having atleast one symptom associated with an ischemic tissue or a tissue damagedby ischemia. The method includes administering to the subject atherapeutically effective amount of a preparation comprising humanhematopoietic stem cell administered a 15-PGDH inhibitor describedherein and/or a therapeutic composition comprising human hematopoieticstem cells and a 15-PGDH inhibitor described herein.

In some embodiments, the ischemia can be associated with at least one ofacute coronary syndrome, acute lung injury (ALI), acute myocardialinfarction (AMI), acute respiratory distress syndrome (ARDS), arterialocclusive disease, arteriosclerosis, articular cartilage defect, asepticsystemic inflammation, atherosclerotic cardiovascular disease,autoimmune disease, bone fracture, bone fracture, brain edema, brainhypoperfusion, Buerger's disease, burns, cancer, cardiovascular disease,cartilage damage, cerebral infarct, cerebral ischemia, cerebral stroke,cerebrovascular disease, chemotherapy-induced neuropathy, chronicinfection, chronic mesenteric ischemia, claudication, congestive heartfailure, connective tissue damage, contusion, coronary artery disease(CAD), critical limb ischemia (CLI), Crohn's disease, deep veinthrombosis, deep wound, delayed ulcer healing, delayed wound-healing,diabetes (type I and type II), diabetic neuropathy, diabetes inducedischemia, disseminated intravascular coagulation (DIC), embolic brainischemia, graft-versus-host disease, hereditary hemorrhagictelengiectasiaischemic vascular disease, hyperoxic injury, hypoxia,inflammation, inflammatory bowel disease, inflammatory disease, injuredtendons, intermittent claudication, intestinal ischemia, ischemia,ischemic brain disease, ischemic heart disease, ischemic peripheralvascular disease, ischemic placenta, ischemic renal disease, ischemicvascular disease, ischemic-reperfusion injury, laceration, left maincoronary artery disease, limb ischemia, lower extremity ischemia,myocardial infarction, myocardial ischemia, organ ischemia,osteoarthritis, osteoporosis, osteosarcoma, Parkinson's disease,peripheral arterial disease (PAD), peripheral artery disease, peripheralischemia, peripheral neuropathy, peripheral vascular disease,pre-cancer, pulmonary edema, pulmonary embolism, remodeling disorder,renal ischemia, retinal ischemia, retinopathy, sepsis, skin ulcers,solid organ transplantation, spinal cord injury, stroke,subchondral-bone cyst, thrombosis, thrombotic brain ischemia, tissueischemia, transient isc hemic attack (TIA), traumatic brain injury,ulcerative colitis, vascular disease of the kidney, vascularinflammatory conditions, von Hippel-Lindau syndrome, and wounds totissues or organs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 (A-C) illustrate graphs showing luciferase activity of cellsthat express a 15-PGDH luciferase fusion construct created by targetedgene knock-in of renilla luciferase into the last coding exon of 15-PGDHtreated with the compounds SW033291, SW054384, and SW145753 at variousconcentrations. The activity is demonstrated in three different coloncancer cell lines all engineered to contain the 15-PGDH-luciferasefusion. These cell lines are Vaco-9m (V9m), LS174T, Vaco503 (V503).

FIG. 2 illustrates western blots demonstrating the levels of 15-PGDHprotein in cell lines V9M, LS174T, and V503 treated with 7.5 μM ofSW033291, SW054384, and SW145753 for 48 hours. Untreated FET cellsprovide a positive control for 15-PGDH expression.

FIGS. 3 (A-C) illustrate western blots demonstrating 15-PGDH proteinlevels in colon cell lines treated with SW124531 (FET cells treated withTGF-β (10 ng/ml for 48 hours) are used as a positive control for 15-PGDHexpression in certain panels).

FIG. 4 illustrates western blots demonstrating the levels of 15-PGDHprotein (wt-PGDH) expressed from a cDNA expression vector inV400-S3-2-32 cells treated with 5 μM SW124531, and protein levels of acatalytically dead mutant 15-PGDH (mu-PGDH) also expressed from a cDNAexpression vector in V400-M3-2-72 cells treated with SW124531.

FIGS. 5 (A-B) illustrate 15-PGDH protein levels in V503 cells treatedwith SW124531 as assayed by immuno-fluorescence (upper two rows) and bywestern blot (lower panel).

FIGS. 6 (A-F) illustrate graphs showing 15-PGDH mRNA levels in coloncancer cell lines treated with SW033291.

FIGS. 7 (A-C) illustrate graphs showing 15-PGDH mRNA levels in coloncancer cell lines treated with SW033291.

FIGS. 8 (A-C) illustrate graphs showing 15-PGDH mRNA levels in coloncancer cell lines treated with SW054384 and SW145753.

FIGS. 9 (A-I) illustrate graphs showing 15-PGDH mRNA levels in coloncancer cell lines treated with 5 μM SW124531.

FIGS. 10 (A-C) illustrate graphs showing 15-PGDH activity in cell linestreated with SW033291, SW054384, and SW145753. Activity is measured aspmol PGE₂/min/million cells.

FIGS. 11 (A-D) illustrates a table and plots showing activity ofrecombinant 15-PGDH protein (a 15-PGDH-GST fusion protein) incubatedwith varying concentrations of the test compounds.

FIGS. 12 (A-D) illustrate plots showing the activity of recombinant15-PGDH protein treated with SW033291 and SW054384, with panels 12A andC measuring transfer of tritium from a radiolabeled PGE2 substrate andpanels 12 B and D measuring generation of NADH by fluorescence.

FIG. 13 illustrates a table and plot showing 15-PGDH activity measuredby following transfer of tritium from a radiolabeled PGE2 substrate incells treated with SW124531 (upper panel) and in recombinant 15-PGDHprotein treated with SW124531 (lower panel).

FIGS. 14 (A-B) illustrate melt curves and a table showing differentcompound's ability to directly bind to recombinant 15-PGDH protein asmeasured by shifting the melting temperature of the protein.

FIGS. 15 (A-B) illustrate melt curves temperature of catalyticallyinactive mutant 15-PGDH protein treated with the test compounds.

FIGS. 16 (A-B) illustrates graphs showing PGE₂ levels that are assayedin the medium of A549 cells that have been stimulated by IL1-beta for 23hours, with the test compounds.

FIG. 17 illustrates a graph showing the dose response effect of SW033291on PGE₂ production from IL1-beta treated A549 cells.

FIGS. 18 (A-B) illustrate graphs showing the in vivo modulations bycompounds (2.5 μM) of PGDH activity as reflected in PGE₂ levelsfollowing addition of PGE₂ into the medium of Vaco-503 cells.

FIG. 19 illustrates images showing the activity of SW033291 in speedingthe healing of a model wound consisting of a scratch in a monolayer ofHaCaT cells observed over 48 hours of treatment.

FIGS. 20 (A-B) illustrate graphs showing the quantitation of scratchwidth at 0 and 48 hours in the control, SW033291 (2.5 μM) treated cells,and the TGF-beta (1 ng/ml) treated cells.

FIGS. 21 (A-B) illustrate plots showing: (A) percent inhibition of PGDHusing titrations of 15-PGDH inhibitor SW033291 run at different 15-PGDHconcentrations; and (B) the IC50 of 15-PGDH inhibitor SW033291 versus15-PGDH concentration.

FIGS. 22 (A-B) indicate that SW033291 behaves very much like anirreversible inhibitor of 15-PGDH, and cannot be efficiently dialyzedoff the 15-PGDH protein.

FIGS. 23 (A-B) illustrate a plot showing reaction rates and relativereaction velocity of 15-PGDH at varying concentrations of SW033291.

FIGS. 24 (A-B) illustrate plots showing: (A) inhibition of 15-PGDH bySW033291 in the presence of PGE-2; and (B) IC₅₀ of SW033291 against15-PGDH versus PGE2 concentration.

FIG. 25 illustrates a schematic diagram showing the structure activityrelationships of analogues of SW033291 versus their IC₅₀ against15-PGDH.

FIG. 26 illustrates a schematic diagram showing additional analogues ofSW033291.

FIGS. 27 (A-C) illustrate graphs showing luciferase activity of coloncancer cell lines V503, LS174T, and V503 treated with 2.5 μM and 7.5 μMthe compounds of FIG. 26 .

FIG. 28 illustrates a graph showing percent inhibition of 15-PGDHactivity by the compounds of FIG. 26 .

FIGS. 29 (A-B) illustrate plots showing the IC₅₀ against 15-PGDH ofSW033291 and SW0206980.

FIGS. 30 (A-B) illustrate plots showing melting profiles of SW0206890binding to 15-PGDH.

FIGS. 31 (A-C) illustrate plots showing percent inhibition of 15-PGDHactivity by SW033291, SW206980, and SW206992.

FIGS. 32 (A-C) illustrate graphs showing luciferase activity of coloncancer cell lines V503, LS174T, and V503 treated with variousconcentrations of SW033291.

FIG. 33 (A-C) illustrate graphs showing luciferase activity of coloncancer cell lines V503, LS174T, and V503 treated with variousconcentrations of SW0206980.

FIG. 34 (A-C) illustrate graphs showing luciferase activity of coloncancer cell lines V503, LS174T, and V503 treated with variousconcentrations of SW0206992.

FIGS. 35 (A-B) illustrate plots showing melting profiles of SW206992,SW0206890 and SW033291 binding to 15-PGDH.

FIGS. 36 (A-B) illustrate plots showing melting profiles of SW206992,SW0206890 and SW033291 binding to 15-PGDH.

FIGS. 37 (A-C) illustrate graphs showing the effect of SW206992,SW0206890 and SW033291 on the regulation of PGE-2 in A549 cellsstimulated with IL1-Beta.

FIGS. 38 (A-C) illustrate graphs showing the effect of SW206992,SW0206890 and SW033291 on cell numbers in A549 cells after stimulatedwith IL1-Beta.

FIG. 39 illustrates a schematic diagram of additional analogues ofSW033291.

FIGS. 40 (A-C) illustrate graphs showing luciferase activity of coloncancer cell lines V9M, LS174T, and V503 treated with 2.5 μM and 7.5 μMthe compounds of FIG. 39 .

FIG. 41 illustrates a graph showing percent inhibition of 15-PGDHactivity by the compounds of FIG. 40 .

FIGS. 42 (A-D) illustrate a graph showing percent inhibition of 15-PGDHactivity by the compounds of FIG. 40 .

FIG. 43 shows the dose response curve for induction of a15-PGDH-luciferase fusion gene reporter in the V9m cell line backgroundof SW033291, SW208064, SW208065, SW208066, and SW208067.

FIG. 44 illustrates titration curves of 15-PGDH inhibitor compounds inan assay measuring effects on PGE2 levels in the medium of A549 cellsthat have been stimulated with IL1-beta.

FIG. 45 is a plot showing weight change of FVB mice treated withSW033291.

FIGS. 46 (A-C) illustrate graphs showing: (A) total bone marrowcellularity; (B) SKL population of wild type versus PGDH^(−/−) mice; and(C) average CFU counts in wild type versus PGDH−/− mice (designated aseither PGDH^(−/−) or as PGDH).

FIG. 47 illustrates a graph showing CFU counts in wild type bone marrowtreated with SW033291 and PGE-2.

FIGS. 48 (A-C) illustrate graphs showing: (A) bone marrow cellularity ofmice treated with SW033291; (B) SKL % in whole bone marrow of micetreated with SW033291; and (C) CFU counts in mice treated SW033291.

FIGS. 49 (A-B) illustrate: (A) a schematic diagram following CD45.2antigen marked cells in lethally irradiated C57BL/6J mice rescued with abone marrow transplant from donor mice treated with SW033291 or withvehicle; and (B) graphs showing chimerism, of donor B-Cells, myeloidcells, and T-Cells after such treatment.

FIG. 50 illustrates a schematic diagram showing schema of a study inwhich C57BL/61 mice are irradiated with 11GY on day 0 and followed bytreatment with SW033291.

FIG. 51 illustrates a schematic diagram of a partial hepatectomy.

FIGS. 52 (A-D) illustrate photographs showing preoperative andpost-operative view of mouse liver.

FIGS. 53 (A-D) illustrate photographs showing post-hepatectomy views ofthe mouse liver (at left) and regeneration of mouse liver onpost-operative day 7 (at right).

FIGS. 54 (A-B) illustrate micrographs of post-hepatectomy mouse liversof mouse administered SW033291 and control vehicle, with arrowsdesignating mitotic figures.

FIG. 55 illustrates a graph showing mitosis in liver of SW033291 treatedmouse versus the control mouse.

FIG. 56 illustrates a graph showing the liver to body weight ratiosattained following partial hepatectomy in control versus SW033291treated C57Bl/6J mice.

FIG. 57 illustrates a graph showing the liver to body weight ratiosattained following partial hepatectomy in control versus SW033291 twicedaily treated C57Bl/6J mice.

FIG. 58 illustrates a graph reprising the liver to body weight ratiosattained following partial hepatectomy in control versus SW033291treated C57Bl/6J mice.

FIGS. 59 (A-B) illustrate a graph and plot showing ALT levels followingpartial hepatectomy in one mouse control versus one mouse treated withSW033291.

FIG. 60 illustrates a graph showing serum bilirubin levels followingpartial hepatectomy in a control mouse and a mouse treated withSW033291.

FIG. 61 illustrates a graph showing the liver to body weight ratiosattained following partial hepatectomy in control versus SW033291treated FVB mice.

FIG. 62 illustrates a graph showing preoperative body weights in controlversus SW033291 treated FVB mice.

FIG. 63 illustrates a graph showing the weight of the resected liversegment from mice treated with either SW033291 or vehicle control andassayed for liver regeneration.

FIG. 64 illustrates a graph showing liver weights attained post partialhepatectomy in SW033291 and control mice.

FIG. 65 illustrates a graph showing the liver to body weight ratiosobtained post partial hepatectomy in SW033291 treated and control mice.

FIG. 66 illustrates a “box and whisker” plot comparing liver to bodyweight ratio following partial hepatectomy of SW033291 treated andcontrol FVB mice at post-operative day 4.

FIG. 67 illustrates a “box and whisker” plot comparing liver to bodyweight ratio following partial hepatectomy of SW033291 treated andcontrol FVB mice at post-operative day 7.

FIG. 68 illustrates a “box and whisker” plot comparing liver to bodyweight ratio following partial hepatectomy of SW033291 treated andcontrol FVB mice at post-operative day 4.

FIG. 69 illustrates photographs of S-phase cells following partialhepatectomy on post-operative day 2 in livers of SW033291 treated andvehicle treated control mice.

FIG. 70 illustrates a photograph showing high powered (40×) views ofrepresentative fields from the study of FIG. 69 .

FIG. 71 illustrates a “box and whiskers” plot comparing percent of BrdUpositive cells in livers of SW033291 treated versus vehicle controltreated mice on post-operative day 2 following partial hepatectomy.

FIG. 72 illustrates a graph showing the average changes from baselineweight of the cohort of control versus SW033291 treated mice all treatedwith 2% dextran sulfate sodium (DSS) in the drinking water.

FIG. 73 illustrates a graph of the daily disease activity index of thecohort of control versus SW033291 treated mice all treated with 2% DSSin the drinking water.

FIG. 74 illustrates a graph showing the average changes from baselineweight of the cohort of DSS treated mice receiving a control vehicleversus SW033291.

FIGS. 75 (A-B) illustrates: (A) a graph showing the number of ulcers ina colon of DSS treated mice receiving a control vehicle versus SW033291;and (B) photographs showing ulcers of DSS treated mice receiving control(left) or SW033291 (right).

FIG. 76 illustrates a graph showing quantitation of ulcer burden on day15 of DSS treated mice receiving a control vehicle or SW033291.

FIGS. 77 (A-B) illustrate photographs showing colonoscopic findings andmouse endoscopic index of colitis severity (MEICs) for a DSS treatedmouse receiving a control vehicle or SW033291.

FIG. 78 illustrates a graph showing MEICS score of DSS treated micereceiving a control vehicle or SW033291.

FIG. 79 illustrates photomicrographs of high powered fields from themid-colon on day 8 of the DSS protocol from control mice, SW033291treated mice (treatment) and 15-PGDH knockout mice (KO) and a graphdepicting sum of the average number of BrdU positive cells per crypt inthe distal plus middle colons of control (Cn), SW033219 treated mice(Tx), and 15-PGDH knockout mice (KO) on day 1, day 8, and day 15 of theDSS treatment protocol.

FIG. 80 illustrates a graph showing colon length at day 22 of DSStreated mice receiving a control vehicle or SW033291.

FIGS. 81 (A-E) illustrate: (A) a schematic illustration showing thedesign of a study of enhanced survival in mice receiving a bone marrowtransplant and also administered the 15-PGDH inhibitor SW033291; (B)graphical survival curves for mice transplanted with 100,000 donorcells; (C) graphical survival curves for mice transplanted with 200,000donor cells; (D) graphical survival curves for mice transplanted with500,000 donor cells; and (E) tabular survival data for all mice in thestudy on study day 30.

FIGS. 82 (A-C) illustrate: (A) a schematic illustration showingmeasurements on blood and bone marrow on day 5 after transplant; (B) agraph showing that SW033291 treated mice have significantly higher totalwhite count; (C) a graph showing that SW033291 treated mice havesignificantly higher total platelet count. The star symbol denotesP<0.05.

FIGS. 83 (A-B) illustrate: (A) a schematic illustration showingmeasurements on blood and bone marrow on day 8 after transplant; and (B)a graph showing that SW033291 treated mice have significantly higherplatelet count than control, with drug treated mice having 77,000platelets compared to control mice having 39,500 platelets. The starsymbol denotes P<0.05.

FIGS. 84 (A-D) illustrate: (A) a schematic illustration showingmeasurements on blood and bone marrow on day 12 after transplant; (B) agraph showing that SW033291 treated mice have significantly higherneutrophil counts, with drug treated mice having 332 neutrophilscompared to control mice having 125 neutrophils; and (C) a graph showingthat on day 12 after transplant, SW033291 treated mice havesignificantly higher hemoglobin count than controls, with drug treatedmice having hemoglobin level of 11.58 and control mice having hemoglobinlevel of 8.3; and D) a graph showing that SW033291 treated mice havesignificantly higher total white counts compared to control mice. Thestar symbol denotes P<0.05.

FIGS. 85 (A-G) illustrate: (A) a schematic illustration showingmeasurements on blood and bone marrow on day 18 after transplant; (B-D)graphs showing SW033291 treated mice have significantly higher totalwhite count (FIG. 85B), lymphocyte count (FIG. 85C), and neutrophilcount (FIG. 85D), with drug treated mice having 835 neutrophils andcontrol mice having 365 neutrophils (FIG. 85D); (E) a graph showing thaton day 18 drug treated mice have significantly higher platelet countsthan control mice; and (F-G) graphs showing drug treated mice havenearly 4-fold increased percentage (FIG. 85F) and total numbers (FIG.85G) of SKL marked bone marrow stem cells than do control mice. The starsymbol denotes P<0.05.

FIGS. 86 (A-B) illustrate graphs showing (A) measurement of PGE2 (pg ofPGE2/mg tissue protein) in 4 different mouse tissues (colon, bonemarrow, liver, lung) across time following IP injection of SW033291 at10 mg/kg; and (B) time course of PGE2 in control mice injected withvehicle only.

FIG. 87 is a schematic illustration showing an experiment in which miceare lethally irradiated (IR) and 12 hours later receive a transplant(BMT) with CFSE dye labeled bone marrow cells (BM), and the number oftransplanted cells that home and survive in the bone marrow of therecipient mice are then determined by FACS at 16 hours post-transplant.

FIG. 88 illustrates a graph showing the percent of CFSE dye labeledcells that have homed to the bone marrow of mice treated as illustratedin FIG. 87 .

FIG. 89 is a schematic illustration showing an experiment in which miceare lethally irradiated (IR) and 12 hours later receive a transplant(BMT) with CFSE dye labeled bone marrow cells (BM), and number oftransplanted cells that home and survive in the bone marrow of therecipient mice are then determined by FACS at 16 hours post-transplant.

FIG. 90 illustrates a graph showing the percent of CFSE dye labeledcells that have homed to the bone marrow of mice treated as illustratedin FIG. 89 .

FIG. 91 is a schematic illustration showing an experiment in which miceare injected with SW033291 twice daily IP at 10 mg/kg for 5 doses.

FIGS. 92 (A-B) illustrate graphs showing induction of gene expression in(A) bone marrow SKL cells and (B) bone marrow stromal cells of SW033291treated mice.

FIG. 93 is a schematic illustration showing an experiment in whichimmune deficient NSG mice are lethally irradiated (IR) and 12 hourslater receive a transplant with CFSE dye labeled buffy coat cells fromhuman umbilical cord blood (UCB), and number of transplanted cells thathome and survive in the bone marrow of the recipient mice are thendetermined by FACS at 16 hours post-transplant.

FIG. 94 illustrates a graph showing the percent of CFSE dye labeledhuman umbilical cord buffy coat cells that have homed to the bone marrowof mice treated as per the schema above.

FIG. 95 illustrates isomers of SW033291 and a representative analyticalHPLC trace.

FIG. 96 illustrates plots showing thermal denaturation of recombinant15-PGDH protein using Differential Scanning Fluorimetry with SYPROOrange.

FIGS. 97 (A-C) illustrate inhibition of recombinant 15-PGDH proteinenzymatic activity by SW033291 stereogenic isomers A and B.

FIG. 98 is a graph showing the activity in inducing activity of a15-PGH-luciferase fusion protein reporter in the Vaco-9M (V9M) cell linebackground of SW033291 isomers A and B.

FIG. 99 is an image showing the conformation of SW209415 docked into theactive site of PGDH.

FIG. 100 illustrates analogs of SW033291.

FIG. 101 illustrates plots of inhibition of 15-PGDH protein by analogsof SW033291 shown in FIG. 100 .

FIG. 102 illustrates a graph showing the dose response effect of analogsof SW033291 shown in FIG. 100 on PGE₂ production from IL1-beta treatedA549 cells.

FIG. 103 illustrates a graph showing luciferase activity of colon cancercell V9m treated with various dosages of analogs of SW033291 shown inFIG. 100 .

FIG. 104 illustrates a graph showing luciferase activity of LS174T cellstreated with various dosages of analogs of SW033291 shown in FIG. 100 .

FIG. 105 illustrates a graph showing luciferase activity of V503 cellstreated with various dosages of analogs of SW033291 shown in FIG. 100 .

FIG. 106 illustrates a graph showing percent inhibition of 15-PGDHactivity by analogs of SW033291 shown in FIG. 100 at 2.5 μM and 7.5 μM.

FIG. 107 illustrates chemical structures of a set of thirteen compounds,designated set 20 with individual compound identifiers ranging fromSW209271 through SW209283, that are structurally related to SW033291.For each compound the molecular weight, tPSA, and C Log P is also shown.

FIGS. 108 and 109 illustrate plots showing the activity of each compoundSW209271 through SW209283 in inhibiting the enzymatic activity ofrecombinant 15-PGDH protein in in vitro assays, with the percentinhibition graphed on the Y-axis and the Log of the compoundconcentration in nM graphed on the X-axis. The IC₅₀ for each compound isrecorded.

FIG. 110 illustrates a graph showing the activity of compounds SW209271through SW209283 in inducing PGE2 as assayed in the medium of A549 cellsthat have first been activated with IL-1beta at concentrations of 4 nM,20 nM, 100 nM, 500 nM, and 2500 nM.

FIG. 111 illustrates a graph showing the activity of compounds SW209271through SW209283 in inducing luciferase activity in a reporter cell lineat concentrations of 19.5 nM, 39.0635 nM, 78.124 nM, 156.25 nM, 312.5nM, 625 nM, and 1250 nM.

FIG. 112 illustrates a graph showing the activity of compounds SW209271through SW209283 in inducing luciferase activity in a reporter cell lineat concentrations of 19.5 nM, 39.0635 nM, 78.124 nM, 156.25 nM, 312.5nM, 625 nM, and 1250 nM.

FIG. 113 illustrates a graph showing the activity of compound SW209271through SW209283 in inducing luciferase activity in a reporter cell lineat concentrations of 19.5 nM, 39.0635 nM, 78.124 nM, 156.25 nM, 312.5nM, 625 nM, and 1250 nM.

FIG. 114 shows chemical structures of two previously describedcompounds, SW209125 and SW208436, along with a set of five compounds,designated set 21 with individual compound identifiers ranging fromSW209239 through SW209333, that are structurally related to SW033291.For each compound the molecular weight, tPSA, and C Log P is also shown.

FIG. 115 illustrates plots showing the activity of compounds SW209125,SW208436 and set 21 compounds ranging from SW209239 through SW209333 ininhibiting the enzymatic activity of recombinant 15-PGDH protein in invitro assays, with the percent inhibition graphed on the Y-axis and theLog of the compound concentration in nM graphed on the X-axis. The IC₅₀for each compound is recorded.

FIG. 116 illustrates chemical structures of a set of six compounds,designated set 23, with individual compound identifiers ranging fromSW209415 through SW209420, that are structurally related to SW033291.For each compound the molecular weight, tPSA, and C Log P is also shown.

FIG. 117 illustrates plots showing the activity of set 23 compoundsranging from SW209415 through SW209420 in inhibiting the enzymaticactivity of recombinant 15-PGDH protein in in vitro assays, with thepercent inhibition graphed on the Y-axis and the Log of the compoundconcentration in nM graphed on the X-axis. The IC₅₀ for each compound isrecorded.

FIG. 118 illustrates a graph showing the activity of selected set 21compounds ranging from SW209239 through SW2093332 and set 23 compoundsranging from SW209415 through SW209420 in inducing PGE2 as assayed inthe medium of A549 cells that have first been activated with IL-1beta atinhibitor concentrations of 4 nM, 20 nM, 100 nM, 500 nM, and 2500 nM.

FIG. 119 illustrates a graph showing the activity of SW209125, SW208436and set 21 compounds ranging from SW209239 through SW209333 in inducingluciferase activity in a reporter cell at concentrations of 19.5 nM,39.0635 nM, 78.124 nM, 156.25 nM, 312.5 nM, 625 nM, and 1250 nM.

FIG. 120 illustrates a graph showing the activity of SW209125, SW208436and set 21 compounds ranging from SW209239 through SW209333 in inducingluciferase activity in a reporter cell line at concentrations of 19.5nM, 39.0635 nM, 78.124 nM, 156.25 nM, 312.5 nM, 625 nM, and 1250 nM.

FIG. 121 illustrates a graph showing the activity of SW209125, SW208436and set 21 compounds ranging from SW209239 through SW209333 in inducingluciferase activity in a reporter cell line at concentrations of 19.5nM, 39.0635 nM, 78.124 nM, 156.25 nM, 312.5 nM, 625 nM, and 1250 nM.

FIG. 122 illustrates a graph showing the activity of set 23 compoundsranging from SW209415 through SW209420 in inducing luciferase activityin a reporter cell line at concentrations of 39.0625 nM, 78.125 nM,156.25 nM, 312.5 nM, 625 nM, 1250 nM, and 2500 nM.

FIG. 123 shows activity of set 23 compounds ranging from SW209415through SW209420 in inducing luciferase activity in a reporter cell lineconstructed from the LS174T colon cancer cell line into which a renillaluciferase cassette has been targeted into the last coding exon of theendogenous 15-PGDH gene to create an in frame fusion gene encoding a15-PGDH-luciferase fusion protein. Activity of SW033291 in the assay isalso shown. Graphed are results of treating the reporter cells with DMSOor with compounds at concentrations of 39.0625 nM, 78.125 nM, 156.25 nM,312.5 nM, 625 nM, 1250 nM, and 2500 nM.

FIG. 124 illustrates a graph showing the activity of set 23 compoundsranging from SW209415 through SW209420 in inducing luciferase activityin a reporter cell line at concentrations of 39.0625 nM, 78.125 nM,156.25 nM, 312.5 nM, 625 nM, 1250 nM, and 2500 nM.

FIG. 125 illustrates the structures of SW209125, set 20 compoundSW209279, and set 23 compounds SW209415 and SW209418.

FIG. 126 illustrates a graph showing the activity of SW209125, set 20compound SW209279, and set 23 compounds SW209415 and SW209418, atconcentrations of 4 nM, 20 nM, 100 nM, 500 nM, and 2500 nM in inducingPGE2 as assayed in the medium of A549 cells that have first beenactivated with IL-1beta.

FIG. 127 illustrates a graph showing the activity of SW209125, set 20compound SW209279, and set 23 compounds SW209415 and SW209418, atconcentrations of 4 nM, 20 nM, 100 nM, 500 nM, and 2500 nM in inducingPGE2 as assayed in the medium of DLD1 cells in media supplemented witharachidonic acid.

FIG. 128 illustrates a graph showing activity of SW033291, SW209125, set20 compound SW209279, and set 23 compounds SW209415 and SW209418, ininducing luciferase activity in a reporter cell line at concentrationsranging from (right to left) of 2.4 nM up to 2500 nM.

FIG. 129 illustrates a graph showing the activity of SW033291, SW209125,set 20 compound SW209279, and set 23 compounds SW209415 and SW209418, ininducing luciferase activity in a reporter cell line at concentrationsranging from (right to left) of 2.4 nM up to 2500 nM.

FIG. 130 illustrates a graph showing the activity of SW033291, SW209125,set 20 compound SW209279, and set 23 compounds SW209415 and SW209418 ininducing luciferase activity in a reporter cell line at concentrationsranging from (right to left) of 2.4 nM up to 2500 nM.

FIG. 131 shows chemical structures of a set of seven compounds,designated set 24, with individual compound identifiers ranging fromSW209427 up to SW209513, that are structurally related to SW033291. Foreach compound the molecular weight, tPSA, and C Log P is also shown.Also shown is the repeated structure of SW209415.

FIG. 132 illustrates plots showing the activity of each compound in set24 with compound numbers from SW209427 up to SW209513 in inhibiting theenzymatic activity of recombinant 15-PGDH protein in in vitro assays,with the percent inhibition graphed on the Y-axis and the Log of thecompound concentration in nM graphed on the X-axis. The IC₅₀ for eachcompound is recorded.

FIG. 133 illustrates a graph showing the activity of set 24 compounds,with compound numbers from SW209427 up to SW209513, in inducingluciferase activity in a reporter cell line at concentrations of 19.5nM, 39.0635 nM, 78.125 nM, 156.25 nM, 312.5 nM, 625 nM, and 1250 nM.

FIG. 134 illustrates a graph showing the activity of set 24 compounds,with compound numbers from SW209427 up to SW209513, in inducingluciferase activity in a reporter cell line at concentrations of 19.5nM, 39.0635 nM, 78.125 nM, 156.25 nM, 312.5 nM, 625 nM, and 1250 nM.

FIGS. 135 (A-C) illustrate dose response curves for inhibition ofenzymatic activity of recombinant 15-PGDH (Y-axis) versus log of the nMconcentration of racemic SW209415 (A), or of the (−) isomer of SW209415(B), or of the (+) isomer of SW209415 (C).

FIG. 136 illustrates the HPLC separation conditions for the enantiomersof SW209415.

FIG. 137 illustrates a graph showing induction of PGE2 that is secretedinto cell culture media of A549 cells that are treated with: DMSO alone;IL1-beta alone; IL1-beta plus racemic SW209415 (labeled SW209415);IL1-beta plus (−) SW209415 (labeled SW209415 (−)); IL1-beta plus (+)SW209415 (labeled SW209415 (+)); or with IL1-beta plus SW033291 (labeledSW033291).

FIGS. 138 (A-D) illustrates graphs showing the measurement of PGE2 (pgof PGE2/mg tissue protein) in 4 different mouse tissues (colon, bonemarrow, liver, lung) across time following IP injection of SW209415 at10 mg/kg.

FIG. 139 illustrates schema of an experiment in which female C57BL/6Jmice are lethally irradiated (IR) and 12 hours later receive atransplant with CFSE dye labeled bone marrow cells from a donor C57BL/6Jfemale mouse, and the number of transplanted cells that home and survivein the bone marrow of the recipient mice are then determined by FACS at16 hours post-transplant.

FIG. 140 illustrates a graph showing the percent of CFSE dye labeleddonor bone marrow cells that have homed to the bone marrow of recipientmice treated as per the schema described in FIG. 139 .

FIG. 141 shows activity of (+) SW209415 in increasing PGE2 in mousetissues.

DETAILED DESCRIPTION

For convenience, certain terms employed in the specification, examples,and appended claims are collected here. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisapplication belongs.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The terms “comprise,” “comprising,” “include,” “including,” “have,” and“having” are used in the inclusive, open sense, meaning that additionalelements may be included. The terms “such as”, “e.g.”, as used hereinare non-limiting and are for illustrative purposes only. “Including” and“including but not limited to” are used interchangeably.

The term “or” as used herein should be understood to mean “and/or”,unless the context clearly indicates otherwise.

As used herein, the term “about” or “approximately” refers to aquantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number,frequency, percentage, dimension, size, amount, weight or length. In oneembodiment, the term “about” or “approximately” refers a range ofquantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%,±2%, or ±1% about a reference quantity, level, value, number, frequency,percentage, dimension, size, amount, weight or length.

It will be noted that the structure of some of the compounds of theapplication include asymmetric (chiral) carbon or sulfur atoms. It is tobe understood accordingly that the isomers arising from such asymmetryare included herein, unless indicated otherwise. Such isomers can beobtained in substantially pure form by classical separation techniquesand by stereochemically controlled synthesis. The compounds of thisapplication may exist in stereoisomeric form, therefore can be producedas individual stereoisomers or as mixtures.

The term “isomerism” means compounds that have identical molecularformulae but that differ in the nature or the sequence of bonding oftheir atoms or in the arrangement of their atoms in space. Isomers thatdiffer in the arrangement of their atoms in space are termed“stereoisomers”. Stereoisomers that are not mirror images of one anotherare termed “diastereoisomers”, and stereoisomers that arenon-superimposable mirror images are termed “enantiomers”, or sometimesoptical isomers. A carbon atom bonded to four nonidentical substituentsis termed a “chiral center” whereas a sulfur bound to three or fourdifferent substitutents, e.g. sulfoxides or sulfinimides, is likewisetermed a “chiral center”.

The term “chiral isomer” means a compound with at least one chiralcenter. It has two enantiomeric forms of opposite chirality and mayexist either as an individual enantiomer or as a mixture of enantiomers.A mixture containing equal amounts of individual enantiomeric forms ofopposite chirality is termed a “racemic mixture”. A compound that hasmore than one chiral center has 2n-1 enantiomeric pairs, where n is thenumber of chiral centers. Compounds with more than one chiral center mayexist as either an individual diastereomer or as a mixture ofdiastereomers, termed a “diastereomeric mixture”. When one chiral centeris present, a stereoisomer may be characterized by the absoluteconfiguration (R or S) of that chiral center. Alternatively, when one ormore chiral centers are present, a stereoisomer may be characterized as(+) or (−). Absolute configuration refers to the arrangement in space ofthe substituents attached to the chiral center. The substituentsattached to the chiral center under consideration are ranked inaccordance with the Sequence Rule of Cahn, Ingold and Prelog. (Cahn etal, Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahn et al.,Angew. Chem. 1966, 78, 413; Cahn and Ingold, J Chem. Soc. 1951 (London),612; Cahn et al., Experientia 1956, 12, 81; Cahn, J., Chem. Educ. 1964,41, 116).

The term “geometric Isomers” means the diastereomers that owe theirexistence to hindered rotation about double bonds. These configurationsare differentiated in their names by the prefixes cis and trans, or Zand E, which indicate that the groups are on the same or opposite sideof the double bond in the molecule according to the Cahn-Ingold-Prelogrules. Further, the structures and other compounds discussed in thisapplication include all atropic isomers thereof.

The term “atropic isomers” are a type of stereoisomer in which the atomsof two isomers are arranged differently in space. Atropic isomers owetheir existence to a restricted rotation caused by hindrance of rotationof large groups about a central bond. Such atropic isomers typicallyexist as a mixture, however as a result of recent advances inchromatography techniques, it has been possible to separate mixtures oftwo atropic isomers in select cases.

The terms “crystal polymorphs” or “polymorphs” or “crystal forms” meanscrystal structures in which a compound (or salt or solvate thereof) cancrystallize in different crystal packing arrangements, all of which havethe same elemental composition. Different crystal forms usually havedifferent X-ray diffraction patterns, infrared spectral, melting points,density hardness, crystal shape, optical and electrical properties,stability and solubility. Recrystallization solvent, rate ofcrystallization, storage temperature, and other factors may cause onecrystal form to dominate. Crystal polymorphs of the compounds can beprepared by crystallization under different conditions.

The term “derivative” refers to compounds that have a common corestructure, and are substituted with various groups as described herein.

The term “bioisostere” refers to a compound resulting from the exchangeof an atom or of a group of atoms with another, broadly similar, atom orgroup of atoms. The objective of a bioisosteric replacement is to createa new compound with similar biological properties to the parentcompound. The bioisosteric replacement may be physicochemically ortopologically based. Examples of carboxylic acid bioisosteres includeacyl sulfonimides, tetrazoles, sulfonates, and phosphonates. See, e.g.,Patani and LaVoie, Chem. Rev. 96, 3147-3176 (1996).

The phrases “parenteral administration” and “administered parenterally”are art-recognized terms, and include modes of administration other thanenteral and topical administration, such as injections, and include,without limitation, intravenous, intramuscular, intrapleural,intravascular, intrapericardial, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular,subarachnoid, intraspinal and intrastemal injection and infusion.

The term “treating” is art-recognized and includes inhibiting a disease,disorder or condition in a subject, e.g., impeding its progress; andrelieving the disease, disorder or condition, e.g., causing regressionof the disease, disorder and/or condition. Treating the disease orcondition includes ameliorating at least one symptom of the particulardisease or condition, even if the underlying pathophysiology is notaffected.

The term “preventing” is art-recognized and includes stopping a disease,disorder or condition from occurring in a subject, which may bepredisposed to the disease, disorder and/or condition but has not yetbeen diagnosed as having it. Preventing a condition related to a diseaseincludes stopping the condition from occurring after the disease hasbeen diagnosed but before the condition has been diagnosed.

The term “pharmaceutical composition” refers to a formulation containingthe disclosed compounds in a form suitable for administration to asubject. In a preferred embodiment, the pharmaceutical composition is inbulk or in unit dosage form. The unit dosage form is any of a variety offorms, including, for example, a capsule, an IV bag, a tablet, a singlepump on an aerosol inhaler, or a vial. The quantity of active ingredient(e.g., a formulation of the disclosed compound or salts thereof) in aunit dose of composition is an effective amount and is varied accordingto the particular treatment involved. One skilled in the art willappreciate that it is sometimes necessary to make routine variations tothe dosage depending on the age and condition of the patient. The dosagewill also depend on the route of administration. A variety of routes arecontemplated, including oral, pulmonary, rectal, parenteral,transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal,intranasal, inhalational, and the like. Dosage forms for the topical ortransdermal administration of a compound described herein includespowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches, nebulized compounds, and inhalants. In a preferred embodiment,the active compound is mixed under sterile conditions with apharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants that are required.

The term “flash dose” refers to compound formulations that are rapidlydispersing dosage forms.

The term “immediate release” is defined as a release of compound from adosage form in a relatively brief period of time, generally up to about60 minutes. The term “modified release” is defined to include delayedrelease, extended release, and pulsed release. The term “pulsed release”is defined as a series of releases of drug from a dosage form. The term“sustained release” or “extended release” is defined as continuousrelease of a compound from a dosage form over a prolonged period.

The phrase “pharmaceutically acceptable” is art-recognized. In certainembodiments, the term includes compositions, polymers and othermaterials and/or dosage forms which are, within the scope of soundmedical judgment, suitable for use in contact with the tissues of humanbeings and animals without excessive toxicity, irritation, allergicresponse, or other problem or complication, commensurate with areasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” is art-recognized, andincludes, for example, pharmaceutically acceptable materials,compositions or vehicles, such as a liquid or solid filler, diluent,excipient, solvent or encapsulating material, involved in carrying ortransporting any subject composition from one organ, or portion of thebody, to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the other ingredientsof a subject composition and not injurious to the patient. In certainembodiments, a pharmaceutically acceptable carrier is non-pyrogenic.Some examples of materials which may serve as pharmaceuticallyacceptable carriers include: (1) sugars, such as lactose, glucose andsucrose; (2) starches, such as corn starch and potato starch; (3)cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5)malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter andsuppository waxes; (9) oils, such as peanut oil, cottonseed oil,sunflower oil, sesame oil, olive oil, corn oil and soybean oil; (10)glycols, such as propylene glycol; (11) polyols, such as glycerin,sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyloleate and ethyl laurate; (13) agar; (14) buffering agents, such asmagnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19)ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxiccompatible substances employed in pharmaceutical formulations.

The compounds of the application are capable of further forming salts.All of these forms are also contemplated herein.

“Pharmaceutically acceptable salt” of a compound means a salt that ispharmaceutically acceptable and that possesses the desiredpharmacological activity of the parent compound. For example, the saltcan be an acid addition salt. One embodiment of an acid addition salt isa hydrochloride salt. The pharmaceutically acceptable salts can besynthesized from a parent compound that contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, non-aqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrilebeing preferred. Lists of salts are found in Remington's PharmaceuticalSciences, 18th ed. (Mack Publishing Company, 1990).

The compounds described herein can also be prepared as esters, forexample pharmaceutically acceptable esters. For example, a carboxylicacid function group in a compound can be converted to its correspondingester, e.g., a methyl, ethyl, or other ester. Also, an alcohol group ina compound can be converted to its corresponding ester, e.g., anacetate, propionate, or other ester.

The compounds described herein can also be prepared as prodrugs, forexample pharmaceutically acceptable prodrugs. The terms “pro-drug” and“prodrug” are used interchangeably herein and refer to any compound,which releases an active parent drug in vivo. Since prodrugs are knownto enhance numerous desirable qualities of pharmaceuticals (e.g.,solubility, bioavailability, manufacturing, etc.) the compounds can bedelivered in prodrug form. Thus, the compounds described herein areintended to cover prodrugs of the presently claimed compounds, methodsof delivering the same and compositions containing the same. “Prodrugs”are intended to include any covalently bonded carriers that release anactive parent drug in vivo when such prodrug is administered to asubject. Prodrugs are prepared by modifying functional groups present inthe compound in such a way that the modifications are cleaved, either inroutine manipulation or in vivo, to the parent compound. Prodrugsinclude compounds wherein a hydroxy, amino, sulfhydryl, carboxy, orcarbonyl group is bonded to any group that may be cleaved in vivo toform a free hydroxyl, free amino, free sulfhydryl, free carboxy or freecarbonyl group, respectively. Prodrugs can also include a precursor(forerunner) of a compound described herein that undergoes chemicalconversion by metabolic processes before becoming an active or moreactive pharmacological agent or active compound described herein.

Examples of prodrugs include, but are not limited to, esters (e.g.,acetate, dialkylaminoacetates, formates, phosphates, sulfates, andbenzoate derivatives) and carbamates (e.g., N,N-dimethylaminocarbonyl)of hydroxy functional groups, ester groups (e.g., ethyl esters,morpholinoethanol esters) of carboxyl functional groups, N-acylderivatives (e.g., N-acetyl) N-Mannich bases, Schiff bases andenaminones of amino functional groups, oximes, acetals, ketals and enolesters of ketone and aldehyde functional groups in compounds, and thelike, as well as sulfides that are oxidized to form sulfoxides orsulfones.

The term “protecting group” refers to a grouping of atoms that whenattached to a reactive group in a molecule masks, reduces or preventsthat reactivity. Examples of protecting groups can be found in Green andWuts, Protective Groups in Organic Chemistry, (Wiley, 2.sup.nd ed.1991); Harrison and Harrison et al., Compendium of Synthetic OrganicMethods, Vols. 1-8 (John Wiley and Sons, 1971-1996); and Kocienski,Protecting Groups, (Verlag, 3^(rd) ed. 2003).

The term “amine protecting group” is intended to mean a functional groupthat converts an amine, amide, or other nitrogen-containing moiety intoa different chemical group that is substantially inert to the conditionsof a particular chemical reaction. Amine protecting groups arepreferably removed easily and selectively in good yield under conditionsthat do not affect other functional groups of the molecule. Examples ofamine protecting groups include, but are not limited to, formyl, acetyl,benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, t-butyloxycarbonyl(Boc), p-methoxybenzyl, methoxymethyl, tosyl, trifluoroacetyl,trimethylsilyl (TMS), fluorenyl-methyloxycarbonyl,2-trimethylsilyl-ethyloxycarbonyl, 1-methyl-1-(4-biphenylyl)ethoxycarbonyl, allyloxycarbonyl, benzyloxycarbonyl (CBZ),2-trimethylsilyl-ethanesulfonyl (SES), trityl and substituted tritylgroups, 9-fluorenylmethyloxycarbonyl (FMOC), nitro-veratryloxycarbonyl(NVOC), and the like. Those of skill in the art can identify othersuitable amine protecting groups.

Representative hydroxy protecting groups include those where the hydroxygroup is either acylated or alkylated such as benzyl, and trityl ethersas well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethersand allyl ethers.

Additionally, the salts of the compounds described herein, can exist ineither hydrated or unhydrated (the anhydrous) form or as solvates withother solvent molecules. Non-limiting examples of hydrates includemonohydrates, dihydrates, etc. Nonlimiting examples of solvates includeethanol solvates, acetone solvates, etc.

The term “solvates” means solvent addition forms that contain eitherstoichiometric or non-stoichiometric amounts of solvent. Some compoundshave a tendency to trap a fixed molar ratio of solvent molecules in thecrystalline solid state, thus forming a solvate. If the solvent is waterthe solvate formed is a hydrate, when the solvent is alcohol, thesolvate formed is an alcoholate. Hydrates are formed by the combinationof one or more molecules of water with one of the substances in whichthe water retains its molecular state as H₂O, such combination beingable to form one or more hydrate.

The compounds, salts and prodrugs described herein can exist in severaltautomeric forms, including the enol and imine form, and the keto andenamine form and geometric isomers and mixtures thereof. Tautomers existas mixtures of a tautomeric set in solution. In solid form, usually onetautomer predominates. Even though one tautomer may be described, thepresent application includes all tautomers of the present compounds. Atautomer is one of two or more structural isomers that exist inequilibrium and are readily converted from one isomeric form to another.This reaction results in the formal migration of a hydrogen atomaccompanied by a switch of adjacent conjugated double bonds. Insolutions where tautomerization is possible, a chemical equilibrium ofthe tautomers will be reached. The exact ratio of the tautomers dependson several factors, including temperature, solvent, and pH. The conceptof tautomers that are interconvertable by tautomerizations is calledtautomerism.

Of the various types of tautomerism that are possible, two are commonlyobserved. In keto-enol tautomerism a simultaneous shift of electrons anda hydrogen atom occurs.

Tautomerizations can be catalyzed by: Base: 1. deprotonation; 2.formation of a delocalized anion (e.g., an enolate); 3. protonation at adifferent position of the anion; Acid: 1. protonation; 2. formation of adelocalized cation; 3. deprotonation at a different position adjacent tothe cation.

The term “analogue” refers to a chemical compound that is structurallysimilar to another but differs slightly in composition (as in thereplacement of one atom by an atom of a different element or in thepresence of a particular functional group, or the replacement of onefunctional group by another functional group). Thus, an analogue is acompound that is similar or comparable in function and appearance, butnot in structure or origin to the reference compound.

A “patient,” “subject,” or “host” to be treated by the subject methodmay mean either a human or non-human animal, such as a mammal, a fish, abird, a reptile, or an amphibian. Thus, the subject of the hereindisclosed methods can be a human, non-human primate, horse, pig, rabbit,dog, sheep, goat, cow, cat, guinea pig or rodent. The term does notdenote a particular age or sex. Thus, adult and newborn subjects, aswell as fetuses, whether male or female, are intended to be covered. Inone aspect, the subject is a mammal. A patient refers to a subjectafflicted with a disease or disorder.

The terms “prophylactic” or “therapeutic” treatment is art-recognizedand includes administration to the host of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic, i.e., it protects thehost against developing the unwanted condition, whereas if it isadministered after manifestation of the unwanted condition, thetreatment is therapeutic (i.e., it is intended to diminish, ameliorate,or stabilize the existing unwanted condition or side effects thereof).

The terms “therapeutic agent”, “drug”, “medicament” and “bioactivesubstance” are art-recognized and include molecules and other agentsthat are biologically, physiologically, or pharmacologically activesubstances that act locally or systemically in a patient or subject totreat a disease or condition. The terms include without limitationpharmaceutically acceptable salts thereof and prodrugs. Such agents maybe acidic, basic, or salts; they may be neutral molecules, polarmolecules, or molecular complexes capable of hydrogen bonding; they maybe prodrugs in the form of ethers, esters, amides and the like that arebiologically activated when administered into a patient or subject.

The phrase “therapeutically effective amount” or “pharmaceuticallyeffective amount” is an art-recognized term. In certain embodiments, theterm refers to an amount of a therapeutic agent that produces somedesired effect at a reasonable benefit/risk ratio applicable to anymedical treatment. In certain embodiments, the term refers to thatamount necessary or sufficient to eliminate, reduce or maintain a targetof a particular therapeutic regimen. The effective amount may varydepending on such factors as the disease or condition being treated, theparticular targeted constructs being administered, the size of thesubject or the severity of the disease or condition. One of ordinaryskill in the art may empirically determine the effective amount of aparticular compound without necessitating undue experimentation. Incertain embodiments, a therapeutically effective amount of a therapeuticagent for in vivo use will likely depend on a number of factors,including: the rate of release of an agent from a polymer matrix, whichwill depend in part on the chemical and physical characteristics of thepolymer; the identity of the agent; the mode and method ofadministration; and any other materials incorporated in the polymermatrix in addition to the agent.

The term “ED50” is art-recognized. In certain embodiments, ED50 meansthe dose of a drug, which produces 50% of its maximum response oreffect, or alternatively, the dose, which produces a pre-determinedresponse in 50% of test subjects or preparations. The term “LD50” isart-recognized. In certain embodiments, LD50 means the dose of a drug,which is lethal in 50% of test subjects. The term “therapeutic index” isan art-recognized term, which refers to the therapeutic index of a drug,defined as LD50/ED50.

The terms “IC₅₀,” or “half maximal inhibitory concentration” is intendedto refer to the concentration of a substance (e.g., a compound or adrug) that is required for 50% inhibition of a biological process, orcomponent of a process, including a protein, subunit, organelle,ribonucleoprotein, etc.

With respect to any chemical compounds, the present application isintended to include all isotopes of atoms occurring in the presentcompounds. Isotopes include those atoms having the same atomic numberbut different mass numbers. By way of general example and withoutlimitation, isotopes of hydrogen include tritium and deuterium, andisotopes of carbon include C-13 and C-14.

When a bond to a substituent is shown to cross a bond connecting twoatoms in a ring, then such substituent can be bonded to any atom in thering. When a substituent is listed without indicating the atom via whichsuch substituent is bonded to the rest of the compound of a givenformula, then such substituent can be bonded via any atom in suchsubstituent. Combinations of substituents and/or variables arepermissible, but only if such combinations result in stable compounds.

When an atom or a chemical moiety is followed by a subscripted numericrange (e.g., C₁₋₆), it is meant to encompass each number within therange as well as all intermediate ranges. For example, “C₁₋₆ alkyl” ismeant to include alkyl groups with 1, 2, 3, 4, 5, 6, 1-6, 1-5, 1-4, 1-3,1-2, 2-6, 2-5, 2-4, 2-3, 3-6, 3-5, 3-4, 4-6, 4-5, and 5-6 carbons.

The term “alkyl” is intended to include both branched (e.g., isopropyl,tert-butyl, isobutyl), straight-chain e.g., methyl, ethyl, propyl,butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl), and cycloalkyl(e.g., alicyclic) groups (e.g., cyclopropyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl), alkyl substituted cycloalkyl groups, andcycloalkyl substituted alkyl groups. Such aliphatic hydrocarbon groupshave a specified number of carbon atoms. For example, C₁₋₆ alkyl isintended to include C₁, C₂, C₃, C₄, C₅, and C₆ alkyl groups. As usedherein, “lower alkyl” refers to alkyl groups having from 1 to 6 carbonatoms in the backbone of the carbon chain. “Alkyl” further includesalkyl groups that have oxygen, nitrogen, sulfur or phosphorous atomsreplacing one or more hydrocarbon backbone carbon atoms. In certainembodiments, a straight chain or branched chain alkyl has six or fewercarbon atoms in its backbone (e.g., C₁-C₆ for straight chain, C₃-C₆ forbranched chain), for example four or fewer. Likewise, certaincycloalkyls have from three to eight carbon atoms in their ringstructure, such as five or six carbons in the ring structure.

The term “substituted alkyls” refers to alkyl moieties havingsubstituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, alkyl,alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkylamino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Cycloalkyls can be further substituted, e.g.,with the substituents described above. An “alkylaryl” or an “aralkyl”moiety is an alkyl substituted with an aryl (e.g., phenylmethyl(benzyl)). If not otherwise indicated, the terms “alkyl” and “loweralkyl” include linear, branched, cyclic, unsubstituted, substituted,and/or heteroatom-containing alkyl or lower alkyl, respectively.

The term “alkenyl” refers to a linear, branched or cyclic hydrocarbongroup of 2 to about 24 carbon atoms containing at least one double bond,such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl,octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, tetracosenyl,cyclopentenyl, cyclohexenyl, cyclooctenyl, and the like. Generally,although again not necessarily, alkenyl groups can contain 2 to about 18carbon atoms, and more particularly 2 to 12 carbon atoms. The term“lower alkenyl” refers to an alkenyl group of 2 to 6 carbon atoms, andthe specific term “cycloalkenyl” intends a cyclic alkenyl group,preferably having 5 to 8 carbon atoms. The term “substituted alkenyl”refers to alkenyl substituted with one or more substituent groups, andthe terms “heteroatom-containing alkenyl” and “heteroalkenyl” refer toalkenyl or heterocycloalkenyl (e.g., heterocylcohexenyl) in which atleast one carbon atom is replaced with a heteroatom. If not otherwiseindicated, the terms “alkenyl” and “lower alkenyl” include linear,branched, cyclic, unsubstituted, substituted, and/orheteroatom-containing alkenyl and lower alkenyl, respectively.

The term “alkynyl” refers to a linear or branched hydrocarbon group of 2to 24 carbon atoms containing at least one triple bond, such as ethynyl,n-propynyl, and the like. Generally, although again not necessarily,alkynyl groups can contain 2 to about 18 carbon atoms, and moreparticularly can contain 2 to 12 carbon atoms. The term “lower alkynyl”intends an alkynyl group of 2 to 6 carbon atoms. The term “substitutedalkynyl” refers to alkynyl substituted with one or more substituentgroups, and the terms “heteroatom-containing alkynyl” and“heteroalkynyl” refer to alkynyl in which at least one carbon atom isreplaced with a heteroatom. If not otherwise indicated, the terms“alkynyl” and “lower alkynyl” include linear, branched, unsubstituted,substituted, and/or heteroatom-containing alkynyl and lower alkynyl,respectively.

The terms “alkyl”, “alkenyl”, and “alkynyl” are intended to includemoieties which are diradicals, i.e., having two points of attachment. Anonlimiting example of such an alkyl moiety that is a diradical is—CH₂CH₂—, i.e., a C₂ alkyl group that is covalently bonded via eachterminal carbon atom to the remainder of the molecule.

The term “alkoxy” refers to an alkyl group bound through a single,terminal ether linkage; that is, an “alkoxy” group may be represented as—O-alkyl where alkyl is as defined above. A “lower alkoxy” group intendsan alkoxy group containing 1 to 6 carbon atoms, and includes, forexample, methoxy, ethoxy, n-propoxy, isopropoxy, t-butyloxy, etc.Preferred substituents identified as “C₁-C₆ alkoxy” or “lower alkoxy”herein contain 1 to 3 carbon atoms, and particularly preferred suchsubstituents contain 1 or 2 carbon atoms (i.e., methoxy and ethoxy).

The term “aryl” refers to an aromatic substituent containing a singlearomatic ring or multiple aromatic rings that are fused together,directly linked, or indirectly linked (such that the different aromaticrings are bound to a common group such as a methylene or ethylenemoiety). Aryl groups can contain 5 to 20 carbon atoms, and particularlypreferred aryl groups can contain 5 to 14 carbon atoms. Examples of arylgroups include benzene, phenyl, pyrrole, furan, thiophene, thiazole,isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole,isooxazole, pyridine, pyrazine, pyridazine, and pyrimidine, and thelike. Furthermore, the term “aryl” includes multicyclic aryl groups,e.g., tricyclic, bicyclic, e.g., naphthalene, benzoxazole,benzodioxazole, benzothiazole, benzoimidazole, benzothiophene,methylenedioxyphenyl, quinoline, isoquinoline, napthridine, indole,benzofuran, purine, benzofuran, deazapurine, or indolizine. Those arylgroups having heteroatoms in the ring structure may also be referred toas “aryl heterocycles”, “heterocycles,” “heteroaryls” or“heteroaromatics”. The aromatic ring can be substituted at one or morering positions with such substituents as described above, as forexample, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl,alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkylamino,dialkylamino, arylamino, diaryl amino, and alkylaryl amino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Aryl groups can also be fused or bridged withalicyclic or heterocyclic rings, which are not aromatic so as to form amulticyclic system (e.g., tetralin, methylenedioxyphenyl). If nototherwise indicated, the term “aryl” includes unsubstituted,substituted, and/or heteroatom-containing aromatic substituents.

The term “alkaryl” refers to an aryl group with an alkyl substituent,and the term “aralkyl” refers to an alkyl group with an arylsubstituent, wherein “aryl” and “alkyl” are as defined above. Exemplaryaralkyl groups contain 6 to 24 carbon atoms, and particularly preferredaralkyl groups contain 6 to 16 carbon atoms. Examples of aralkyl groupsinclude, without limitation, benzyl, 2-phenyl-ethyl, 3-phenyl-propyl,4-phenyl-butyl, 5-phenyl-pentyl, 4-phenylcyclohexyl, 4-benzylcyclohexyl,4-phenylcyclohexylmethyl, 4-benzylcyclohexylmethyl, and the like.Alkaryl groups include, for example, p-methylphenyl, 2,4-dimethylphenyl,p-cyclohexylphenyl, 2,7-dimethylnaphthyl, 7-cyclooctylnaphthyl,3-ethyl-cyclopenta-1,4-diene, and the like.

The terms “heterocyclyl” or “heterocyclic group” include closed ringstructures, e.g., 3- to 10-, or 4- to 7-membered rings, which includeone or more heteroatoms. “Heteroatom” includes atoms of any elementother than carbon or hydrogen. Examples of heteroatoms include nitrogen,oxygen, sulfur and phosphorus.

Heterocyclyl groups can be saturated or unsaturated and includepyrrolidine, oxolane, thiolane, piperidine, piperazine, morpholine,lactones, lactams, such as azetidinones and pyrrolidinones, sultams, andsultones. Heterocyclic groups such as pyrrole and furan can havearomatic character. They include fused ring structures, such asquinoline and isoquinoline. Other examples of heterocyclic groupsinclude pyridine and purine. The heterocyclic ring can be substituted atone or more positions with such substituents as described above, as forexample, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl,cyano, azido, heterocyclyl, or an aromatic or heteroaromatic moiety.Heterocyclic groups can also be substituted at one or more constituentatoms with, for example, a lower alkyl, a lower alkenyl, a lower alkoxy,a lower alkylthio, a lower alkylamino, a lower alkylcarboxyl, a nitro, ahydroxyl, —CF₃, or —CN, or the like.

The term “halo” or “halogen” refers to fluoro, chloro, bromo, and iodo.“Counterion” is used to represent a small, negatively charged speciessuch as fluoride, chloride, bromide, iodide, hydroxide, acetate, andsulfate. The term sulfoxide refers to a sulfur attached to 2 differentcarbon atoms and one oxygen and the S—O bond can be graphicallyrepresented with a double bond (S═O), a single bond without charges(S—O) or a single bond with charges [S(+)—O(−)].

The terms “substituted” as in “substituted alkyl,” “substituted aryl,”and the like, as alluded to in some of the aforementioned definitions,is meant that in the alkyl, aryl, or other moiety, at least one hydrogenatom bound to a carbon (or other) atom is replaced with one or morenon-hydrogen substituents. Examples of such substituents include,without limitation: functional groups such as halo, hydroxyl, silyl,sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀aryloxy, acyl (including C₂-C₂₄ alkylcarbonyl (—CO-alkyl) and C₆-C₂₀arylcarbonyl (—CO-aryl)), acyloxy (—O-acyl), C₂-C₂₄ alkoxycarbonyl(—(CO)—O-alkyl), C₆-C₂₀ aryloxycarbonyl (—(CO)—O-aryl), C₂-C₂₄alkylcarbonato (—O—(CO)—O-alkyl), C₆-C₂₀ arylcarbonato (—O—(CO)—O-aryl),carboxy (—COOH), carboxylato (—COO—), carbamoyl (—(CO)—NH₂),mono-(C₁-C₂₄ alkyl)-substituted carbamoyl (—(CO)—NH(C₁-C₂₄ alkyl)),di-(C₁-C₄ alkyl)-substituted carbamoyl (—(CO)—N(C₁-C₂₄ alkyl)₂),mono-substituted arylcarbamoyl (—(CO)—NH-aryl), thiocarbamoyl(—(CS)—NH₂), carbamido (—NH—(CO)—NH₂), cyano (—CN), isocyano (—N⁺C⁻),cyanato (—O—CN), isocyanato (—ON⁺C⁻), isothiocyanato (—S—CN), azido(—N═N⁺═N⁻), formyl (—(CO)—H), thioformyl (—(CS)—H), amino (—NH₂), mono-and di-(C₁-C₂₄ alkyl)-substituted amino, mono- and di-(C₅-C₂₀aryl)-substituted amino, C₂-C₂₄ alkylamido (—NH—(CO)-alkyl), C₆-C₂₀arylamido (—NH—(CO)-aryl), imino (—CR═NH where R=hydrogen, C₁-C₂₄ alkyl,C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, etc.), alkylimino(—CR═N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), arylimino(—CR═N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro(—NO₂), nitroso (—NO), sulfo (—SO₂—OH), sulfonato (—SO₂—O⁻), C₁-C₂₄alkylsulfanyl (—S-alkyl; also termed “alkylthio”), arylsulfanyl(—S-aryl; also termed “arylthio”), C₁-C₂₄ alkylsulfinyl (—(SO)-alkyl),C₅-C₂₀ arylsulfinyl (—(SO)-aryl), C₁-C₂₄ alkylsulfonyl (—SO₂-alkyl),C₅-C₂₀ arylsulfonyl (—SO₂-aryl), phosphono (—P(O)(OH)₂), phosphonato(—P(O)(O⁻)₂), phosphinato (—P(O)(O⁻)), phospho (—PO₂), and phosphino(—PH₂); and the hydrocarbyl moieties C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl,C₂-C₂₄ alkynyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, and C₆-C₂₄ aralkyl.

In addition, the aforementioned functional groups may, if a particulargroup permits, be further substituted with one or more additionalfunctional groups or with one or more hydrocarbyl moieties such as thosespecifically enumerated above. Analogously, the above-mentionedhydrocarbyl moieties may be further substituted with one or morefunctional groups or additional hydrocarbyl moieties such as thosespecifically enumerated.

When the term “substituted” appears prior to a list of possiblesubstituted groups, it is intended that the term apply to every memberof that group. For example, the phrase “substituted alkyl, alkenyl, andaryl” is to be interpreted as “substituted alkyl, substituted alkenyl,and substituted aryl.” Analogously, when the term“heteroatom-containing” appears prior to a list of possibleheteroatom-containing groups, it is intended that the term apply toevery member of that group. For example, the phrase“heteroatom-containing alkyl, alkenyl, and aryl” is to be interpreted as“heteroatom-containing alkyl, substituted alkenyl, and substituted aryl.

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.For example, the phrase “optionally substituted” means that anon-hydrogen substituent may or may not be present on a given atom, and,thus, the description includes structures wherein a non-hydrogensubstituent is present and structures wherein a non-hydrogen substituentis not present.

The terms “stable compound” and “stable structure” are meant to indicatea compound that is sufficiently robust to survive isolation, and asappropriate, purification from a reaction mixture, and formulation intoan efficacious therapeutic agent.

The terms “free compound” is used herein to describe a compound in theunbound state.

Throughout the description, where compositions are described as having,including, or comprising, specific components, it is contemplated thatcompositions also consist essentially of, or consist of, the recitedcomponents. Similarly, where methods or processes are described ashaving, including, or comprising specific process steps, the processesalso consist essentially of, or consist of, the recited processingsteps. Further, it should be understood that the order of steps or orderfor performing certain actions is immaterial so long as the compositionsand methods described herein remains operable. Moreover, two or moresteps or actions can be conducted simultaneously.

The term “small molecule” is an art-recognized term. In certainembodiments, this term refers to a molecule, which has a molecularweight of less than about 2000 amu, or less than about 1000 amu, andeven less than about 500 amu.

All percentages and ratios used herein, unless otherwise indicated, areby weight.

The term “neoplasm” refers to any abnormal mass of cells or tissue as aresult of neoplasia. The neoplasm may be benign, potentially malignant(precancerous), or malignant (cancerous). An adenoma is an example of aneoplasm.

The terms “adenoma”, “colon adenoma” and “polyp” are used herein todescribe any precancerous neoplasm of the colon.

The term “colon” as used herein is intended to encompass the right colon(including the cecum), the transverse colon, the left colon and therectum.

The terms “colorectal cancer” and “colon cancer” are usedinterchangeably herein to refer to any cancerous neoplasia of the colon(including the rectum, as defined above).

The terms “gene expression” or “protein expression” includes anyinformation pertaining to the amount of gene transcript or proteinpresent in a sample, as well as information about the rate at whichgenes or proteins are produced or are accumulating or being degraded(e.g., reporter gene data, data from nuclear runoff experiments,pulse-chase data etc.). Certain kinds of data might be viewed asrelating to both gene and protein expression. For example, proteinlevels in a cell are reflective of the level of protein as well as thelevel of transcription, and such data is intended to be included by thephrase “gene or protein expression information”. Such information may begiven in the form of amounts per cell, amounts relative to a controlgene or protein, in unitless measures, etc.; the term “information” isnot to be limited to any particular means of representation and isintended to mean any representation that provides relevant information.The term “expression levels” refers to a quantity reflected in orderivable from the gene or protein expression data, whether the data isdirected to gene transcript accumulation or protein accumulation orprotein synthesis rates, etc.

The terms “healthy” and “normal” are used interchangeably herein torefer to a subject or particular cell or tissue that is devoid (at leastto the limit of detection) of a disease condition.

The term “nucleic acid” refers to polynucleotides such asdeoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include analogues of eitherRNA or DNA made from nucleotide analogues, and, as applicable to theembodiment being described, single-stranded (such as sense or antisense)and double-stranded polynucleotides. In some embodiments, “nucleic acid”refers to inhibitory nucleic acids. Some categories of inhibitorynucleic acid compounds include antisense nucleic acids, RNAi constructs,and catalytic nucleic acid constructs. Such categories of nucleic acidsare well-known in the art.

Embodiments described herein relate to compounds and methods ofmodulating SCD activity (e.g., 15-PGDH activity), modulating tissueprostaglandin levels, and/or treating diseases, disorders, or conditionsin which it is desired to modulate 15-PGDH activity and/or prostaglandinlevels.

“Inhibitors,” “activators,” and “modulators” of 15-PGDH expression or of15-PGDH activity are used to refer to inhibitory, activating, ormodulating molecules, respectively, identified using in vitro and invivo assays for 15-PGDH expression or 15-PGDH activity, e.g., ligands,agonists, antagonists, and their homologs and mimetics. The term“modulator” includes inhibitors and activators. Inhibitors are agentsthat, e.g., inhibit expression of 15-PGDH or bind to, partially ortotally block stimulation, decrease, prevent, delay activation,inactivate, desensitize, or down regulate the activity of 15-PGDH, e.g.,antagonists. Activators are agents that, e.g., induce or activate theexpression of a 15-PGDH or bind to, stimulate, stabilize, increase,open, activate, facilitate, or enhance activation, sensitize or upregulate the activity of 15-PGDH, e.g., agonists. Modulators includenaturally occurring and synthetic ligands, small chemical molecules, andthe like.

15-PGDH inhibitors described herein can provide a pharmacologic methodfor elevating prostaglandin levels in tissue. Known activities ofprostaglandins include promoting hair growth, promoting skinpigmentation, and promoting skin darkening or the appearance of skintanning. Known activities of prostaglandins also include amelioratingpulmonary artery hypertension. 15-PGDH inhibitors described herein mayalso be utilized to increase tissue stem cell numbers for purposes thatwould include increasing resistance to tissue damage by radiation,increasing resistance to environmental exposures to radiation,increasing stem cell numbers to increase fitness of bone marrow or othertypes of transplantation (through either in vivo exposure to 15-PGDHinhibitors described herein to increase stem cell numbers prior toharvest of a transplanted tissue, or through ex vivo exposure of aharvested tissue prior to transplant into a recipient host, or throughtreatment of the graft recipient). 15-PGDH inhibitors described hereinmay also be utilized for purposes that would include promoting liverregeneration, including liver regeneration after liver resection, andliver regeneration after toxic insults, which for example may be thetoxic insult of acetaminophen overdose. Prostaglandin signaling is alsoknown to promote wound healing, protect the stomach from ulceration, andpromote healing of ulcers of stomach and intestines. Additionally,15-PGDH inhibitors described herein can promote activity of humankeratinocytes in “healing” scratches across cultures of keratinocytecells. Hence, 15-PGDH inhibitors described herein may be utilized toalso heal ulcers of other tissues, including, but not limited to skin,and including but not limited to diabetic ulcers. Further, 15-PGDHinhibitors described herein may be utilized for the treatment oferectile dysfunction.

15-PGDH activators described herein can increase levels of 15-PGDHprotein in cells and in increase levels of 15-PGDH enzymatic activity incells. Increasing tissue levels of 15-PGDH can decrease tissue levels ofprostaglandins. Activities associated with compounds that decreasetissue prostaglandins include decreasing development of human tumors,particularly decreasing development of human colon tumors. Accordingly,compounds that increase tissue 15-PGDH activity can lower risk ofdevelopment of colon and other tumors. Compounds that increase 15-PGDHactivity can also be used to treat colon and other tumors. Compoundsthat increase 15-PGDH may be used to treat or to prevent tumors whengiven singly, or when given in combination with inhibitors ofcyclooxygenase-1 and/or cyclooxygenase-2 enzymes, or when given incombination with other therapeutic agents.

15-PGDH inhibitors and activators described herein can be identifiedusing assays in which putative modulator compounds are applied to cellsexpressing 15-PGDH and then the functional effects on 15-PGDH activityare determined. Samples or assays comprising 15-PGDH that are treatedwith a potential activator, inhibitor, or modulator are compared tocontrol samples without the inhibitor, activator, or modulator toexamine the extent of effect. Control samples (untreated withmodulators) are assigned a relative 15-PGDH activity value of 100%.Inhibition of 15-PGDH is achieved when the 15-PGDH activity valuerelative to the control is about 80%, optionally 50% or 25%, 10%, 5% or1%. Activation of 15-PGDH is achieved when the 15-PGDH activity orexpression value relative to the control is 105%, optionally 110%,optionally 125%, optionally 150%, optionally 200%, 300%, 400%, 500%, or1000-3000% or more higher.

Agents tested as modulators of SCD (e.g., 15-PGDH) can be any smallchemical molecule or compound. Typically, test compounds will be smallchemical molecules, natural products, or peptides. The assays aredesigned to screen large chemical libraries by automating the assaysteps and providing compounds from any convenient source to assays,which are typically run in parallel (e.g., in microtiter formats onmicrotiter plates in robotic assays). Modulators also include agentsdesigned to increase the level of 15-PGDH mRNA or the level oftranslation from an mRNA.

In some embodiments, the modulator of SCD (e.g., 15-PGDH) can be a15-PGDH inhibitor that includes a compound having the following formula(V):

-   -   wherein n=0-2;    -   R₁ and R₃ are the same or different and are each selected from        the group consisting of:

-   -   R₂ is N or CR₇;    -   R₄ is selected from the group consisting of H, Cl, F, NH₂, and        N(R₆)₂;    -   R₅ is selected from the group consisting of branched or linear        alkyl including —(CH₂)n₁CH₃ (n₁=0-7),

wherein n₂=0-6 and X is any of the following: CF_(y)H_(z) (y+z=3),CCl_(y)H_(z) (y+z=3), OH, OAc, OMe, R₆, OR₆, CN, N(R₆)₂,

(n₃=0-5, m=1-5), and

(n₄=0-5);

-   -   each R₆ and R₇ are the same or different and are one or more        substituent selected from the group consisting of hydrogen,        substituted or unsubstituted C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl,        C₂-C₂₄ alkynyl, C₃-C₂₀ aryl, heterocycloalkenyl containing from        5-6 ring atoms, (wherein from 1-3 of the ring atoms is        independently selected from N, NH, N(C₁-C₆ alkyl), NC(O)(C₁-C₆        alkyl), O, and S), heteroaryl or heterocyclyl containing from        5-14 ring atoms, (wherein from 1-6 of the ring atoms is        independently selected from N, NH, N(C₁-C₃ alkyl), O, and S),        C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, halo, silyl, hydroxyl,        sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy,        C₅-C₂₀ aryloxy, acyl (including C₂-C₂₄ alkylcarbonyl (—CO-alkyl)        and C₆-C₂₀ arylcarbonyl (—CO-aryl)), acyloxy (—O-acyl), C₂-C₂₄        alkoxycarbonyl (—(CO)—O-alkyl), C₆-C₂₀ aryloxycarbonyl        (—(CO)—O-aryl), C₂-C₂₄ alkylcarbonato (—O—(CO)—O-alkyl), C₆-C₂₀        arylcarbonato (—O—(CO)—O-aryl), carboxy (—COOH), carboxylato        (—COO⁻), carbamoyl (—(CO)—NH₂), C₁-C₂₄ alkyl-carbamoyl        (—(CO)—NH(C₁-C₂₄ alkyl)), arylcarbamoyl (—(CO)—NH-aryl),        thiocarbamoyl (—(CS)—NH₂), carbamido (—NH—(CO)—NH₂), cyano        (—CN), isocyano (—N⁺C⁻), cyanato (—O—CN), isocyanato (—O—N⁺═C⁻),        isothiocyanato (—S—CN), azido (—N═N⁺═N⁻), formyl (—(CO)—H),        thioformyl (—(CS)—H), amino (—NH₂), C₁-C₂₄ alkyl amino, C₅-C₂₀        aryl amino, C₂-C₂₄ alkylamido (—NH—(CO)-alkyl), C₆-C₂₀ arylamido        (—NH—(CO)-aryl), sulfanamido (—SO2NR2 where R is independently        H, alkyl, aryl or heteroaryl), imino (—CR═NH where R is        hydrogen, C₁-C₂₄ alkyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄        aralkyl, etc.), alkylimino (—CR═N(alkyl), where R=hydrogen,        alkyl, aryl, alkaryl, aralkyl, etc.), arylimino (—CR═N(aryl),        where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (—NO₂),        nitroso (—NO), sulfo (—SO₂—OH), sulfonato (—SO₂—O⁻), C₁-C₂₄        alkylsulfanyl (—S-alkyl; also termed “alkylthio”), arylsulfanyl        (—S-aryl; also termed “arylthio”), C₁-C₂₄ alkylsulfinyl        (—(SO)-alkyl), C₅-C₂₀ arylsulfinyl (—(SO)-aryl), C₁-C₂₄        alkylsulfonyl (—SO₂-alkyl), C₅-C₂₀ arylsulfonyl (—SO₂-aryl),        sulfonamide (—SO₂—NH2, —SO₂NY₂ (wherein Y is independently H,        aryl or alkyl), phosphono (—P(O)(OH)₂), phosphonato        (—P(O)(O⁻)₂), phosphinato (—P(O)(O⁻)), phospho (—PO₂), phosphino        (—PH₂), polyalkyl ethers (—[(CH₂)_(n)O]_(m)), phosphates,        phosphate esters [—OP(O)(OR)₂ where R=H, methyl or other alkyl],        groups incorporating amino acids or other moieties expected to        bear positive or negative charge at physiological pH, and        combinations thereof;    -   R₃ is not hydrogen if R₁ is H, an unsubstituted thiophene, or an        unsubstituted thiazole and R₅ is butyl; or R₃ is not an        unsubstituted phenyl if R₁ is H, or an unsubstituted phenyl,        thiophene, or thiazole and R₅ is benzyl or (CH₂)n₅(CH₃)        (n₅=0-5); and pharmaceutically acceptable salts thereof.

In some embodiments, R₂ can be N or CH. R₁ can be a substituted orunsubstituted heterocyclyl containing 5-6 ring atoms. For example, R₁can be a substituted or unsubstituted thiophene, thiazole, oxazole,imidazole, pyridine, or phenyl. R₃ can be selected from the groupconsisting of H, substituted or unsubstituted aryl, a substituted orunsubstituted cycloalkyl, and a substituted or unsubstitutedheterocyclyl, alkyl, or carboxy including carboxylic acid (—CO2H),carboxy ester (—CO₂alkyl) and carboxamide [—CON(H)(alkyl) or—CO₂N(alkyl)₂].

In still other embodiments, 15-PGDH inhibitor can include a compoundhaving formula (V₁)

-   -   wherein n=0-2;    -   R₃ is selected from the group consisting of:

-   -   R₂ is N or CR₇;    -   R₄ is selected from the group consisting of H, Cl, F, NH₂, and        N(R₆)₂;    -   R₅ is selected from the group consisting of branched or linear        alkyl including —(CH₂)n₁CH₃ (n₁=0-7),

wherein n₂=0-6 and X is any of the following: CF_(y)H_(z) (y+z=3),CCl_(y)H_(z) (y+z=3), OH, OAc, OMe, R₆, OR₆, CN, N(R₆)₂,

(n₃=0-5, m=1-5), and

(n₄=0-5);

-   -   each R₆ and R₇ are the same or different and are one or more        substituent selected from the group consisting of hydrogen,        substituted or unsubstituted C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl,        C₂-C₂₄ alkynyl, C₃-C₂₀ aryl, heterocycloalkenyl containing from        5-6 ring atoms, (wherein from 1-3 of the ring atoms is        independently selected from N, NH, N(C₁-C₆ alkyl), NC(O)(C₁-C₆        alkyl), O, and S), heteroaryl containing from 5-14 ring atoms,        (wherein from 1-6 of the ring atoms is independently selected        from N, NH, N(C₁-C₃ alkyl), O, and S), C₆-C₂₄ alkaryl, C₆-C₂₄        aralkyl, halo, silyl, hydroxyl, sulfhydryl, C₁-C₂₄ alkoxy,        C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀ aryloxy, acyl        (including C₂-C₂₄ alkylcarbonyl (—CO-alkyl) and C₆-C₂₀        arylcarbonyl (—CO-aryl)), acyloxy (—O-acyl), C₂-C₂₄        alkoxycarbonyl (—(CO)—O-alkyl), C₆-C₂₀ aryloxycarbonyl        (—(CO)—O-aryl), C₂-C₂₄ alkylcarbonato (—O—(CO)—O-alkyl), C₆-C₂₀        arylcarbonato (—O—(CO)—O-aryl), carboxy (—COOH), carboxylato        (—COO⁻), carbamoyl (—(CO)—NH₂), C₁-C₂₄ alkyl-carbamoyl        (—(CO)—NH(C₁-C₂₄ alkyl)), arylcarbamoyl (—(CO)—NH-aryl),        thiocarbamoyl (—(CS)—NH₂), carbamido (—NH—(CO)—NH₂), cyano        (—CN), isocyano (—N⁺C⁻), cyanato (—O—CN), isocyanato (—O—N⁺═C⁻),        isothiocyanato (—S—CN), azido (—N═N⁺═N⁻), formyl (—(CO)—H),        thioformyl (—(CS)—H), amino (—NH₂), C₁-C₂₄ alkyl amino, C₅-C₂₀        aryl amino, C₂-C₂₄ alkylamido (—NH—(CO)-alkyl), C₆-C₂₀ arylamido        (—NH—(CO)-aryl), sulfanamido (—SO2NR2 where R is independently        H, alkyl, aryl or heteroaryl), imino (—CR═NH where R is        hydrogen, C₁-C₂₄ alkyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄        aralkyl, etc.), alkylimino (—CR═N(alkyl), where R=hydrogen,        alkyl, aryl, alkaryl, aralkyl, etc.), arylimino (—CR═N(aryl),        where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (—NO₂),        nitroso (—NO), sulfo (—SO₂—OH), sulfonato (—SO₂—O⁻), C₁-C₂₄        alkylsulfanyl (—S-alkyl; also termed “alkylthio”), arylsulfanyl        (—S-aryl; also termed “arylthio”), C₁-C₂₄ alkylsulfinyl        (—(SO)-alkyl), C₅-C₂₀ arylsulfinyl (—(SO)-aryl), C₁-C₂₄        alkylsulfonyl (—SO₂-alkyl), C₅-C₂₀ arylsulfonyl (—SO₂-aryl),        sulfonamide (—SO₂—NH2, —SO₂NY₂ (wherein Y is independently H,        aryl or alkyl), phosphono (—P(O)(OH)₂), phosphonato        (—P(O)(O⁻)₂), phosphinato (—P(O)(O⁻)), phospho (—PO₂), phosphino        (—PH₂), polyalkyl ethers (—[(CH₂)_(n)O]_(m)), phosphates,        phosphate esters [—OP(O)(OR)₂ where R=H, methyl or other alkyl],        groups incorporating amino acids or other moieties expected to        bear positive or negative charge at physiological pH, and        combinations thereof;    -   R₃ is not hydrogen if R₅ is butyl; or R₃ is not an unsubstituted        phenyl if R₅ is benzyl or (CH₂)n₅(CH₃) (n₅=0-5); and        pharmaceutically acceptable salts thereof.

Examples of 15-PGDH inhibitors having formula (V) include the followingcompounds:

-   -   and pharmaceutically acceptable salts thereof.

In still other embodiments, R⁶ and R⁷ can independently be a group thatimproves aqueous solubility, for example, a phosphate ester (—OPO₃H₂), aphenyl ring linked to a phosphate ester (—OPO₃H₂), a phenyl ringsubstituted with one or more methoxyethoxy groups, or a morpholine, oran aryl or heteroaryl ring substituted with such a group.

In other embodiments, the 15-PGDH inhibitor can include a compoundhaving the following formula (X):

-   -   wherein n is 0-2    -   R¹ and R¹¹ are the same or different and are one or more        substituent selected from the group consisting of hydrogen,        substituted or unsubstituted C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl,        C₂-C₂₄ alkynyl, C₃-C₂₀ aryl, heterocycloalkenyl containing from        5-6 ring atoms, (wherein from 1-3 of the ring atoms is        independently selected from N, NH, N(C₁-C₆ alkyl), NC(O)(C₁-C₆        alkyl), O, and S), heteroaryl containing from 5-14 ring atoms,        (wherein from 1-6 of the ring atoms is independently selected        from N, NH, N(C₁-C₃ alkyl), O, and S), C₆-C₂₄ alkaryl, C₆-C₂₄        aralkyl, halo, silyl, hydroxyl, sulfhydryl, C₁-C₂₄ alkoxy,        C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀ aryloxy, acyl        (including C₂-C₂₄ alkylcarbonyl (—CO-alkyl) and C₆-C₂₀        arylcarbonyl (—CO-aryl)), acyloxy (—O-acyl), C₂-C₂₄        alkoxycarbonyl (—(CO)—O-alkyl), C₆-C₂₀ aryloxycarbonyl        (—(CO)—O-aryl), C₂-C₂₄ alkylcarbonato (—O—(CO)—O-alkyl), C₆-C₂₀        arylcarbonato (—O—(CO)—O-aryl), carboxy (—COOH), carboxylato        (—COO⁻), carbamoyl (—(CO)—NH₂), C₁-C₂₄ alkyl-carbamoyl        (—(CO)—NH(C₁-C₂₄ alkyl)), arylcarbamoyl (—(CO)—NH-aryl),        thiocarbamoyl (—(CS)—NH₂), carbamido (—NH—(CO)—NH₂), cyano        (—CN), isocyano (—N⁺C⁻), cyanato (—O—CN), isocyanato (—O—N⁺═C⁻),        isothiocyanato (—S—CN), azido (—N═N⁺═N⁻), formyl (—(CO)—H),        thioformyl (—(CS)—H), amino (—NH₂), C₁-C₂₄ alkyl amino, C₅-C₂₀        aryl amino, C₂-C₂₄ alkylamido (—NH—(CO)-alkyl), C₆-C₂₀ arylamido        (—NH—(CO)-aryl), sulfanamido (—SO2NR2 where R is independently        H, alkyl, aryl or heteroaryl), imino (—CR═NH where R is        hydrogen, C₁-C₂₄ alkyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄        aralkyl, etc.), alkylimino (—CR═N(alkyl), where R=hydrogen,        alkyl, aryl, alkaryl, aralkyl, etc.), arylimino (—CR═N(aryl),        where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (—NO₂),        nitroso (—NO), sulfo (—SO₂—OH), sulfonato (—SO₂—O⁻), C₁-C₂₄        alkylsulfanyl (—S-alkyl; also termed “alkylthio”), arylsulfanyl        (—S-aryl; also termed “arylthio”), C₁-C₂₄ alkylsulfinyl        (—(SO)-alkyl), C₅-C₂₀ arylsulfinyl (—(SO)-aryl), C₁-C₂₄        alkylsulfonyl (—SO₂-alkyl), C₅-C₂₀ arylsulfonyl (—SO₂-aryl),        sulfonamide (—SO₂—NH2, —SO₂NY₂ (wherein Y is independently H,        aryl or alkyl), phosphono (—P(O)(OH)₂), phosphonato        (—P(O)(O⁻)₂), phosphinato (—P(O)(O⁻)), phospho (—PO₂), phosphino        (—PH₂), polyalkyl ethers (—[(CH₂)_(n)O]_(m)), phosphates,        phosphate esters [—OP(O)(OR)₂ where R=H, methyl or other alkyl],        groups incorporating amino acids or other moieties expected to        bear positive or negative charge at physiological pH, and        combinations thereof;    -   X¹⁰, X¹¹, X¹², X¹³, and X¹⁴ are independently N or CR^(c),        wherein R^(c) is H or a C₁₋₈ alkyl, which is linear, branched,        or cyclic, and which is unsubstituted or substituted, and        wherein at least one of X¹¹, X¹², X¹³, and X¹⁴ is CR^(c);    -   Z² is O, S, CR^(a)R^(b) or NR^(a), wherein R^(a) and R^(b) are        independently H or a C₁₋₈ alkyl, which is linear, branched, or        cyclic, and which is unsubstituted or substituted;    -   and pharmaceutically acceptable salts thereof.

Examples of 15-PGDH inhibitors having formulas (X) include the followingcompounds:

and pharmaceutically acceptable salts thereof.

In certain embodiments, the 15-PGDH inhibitor having formula (V), (V₁),and (X) can be selected that can ia) at 2.5 μM concentration, stimulatea Vaco503 reporter cell line expressing a 15-PGDH luciferase fusionconstruct to a luciferase output level of greater than 70 (using a scaleon which a value of 100 indicates a doubling of reporter output overbaseline); iia) at 2.5 μM concentration stimulate a V9m reporter cellline expressing a 15-PGDH luciferase fusion construct to a luciferaseoutput level of greater than 75; iiia) at 7.5 μM concentration stimulatea LS174T reporter cell line expressing a 15-PGDH luciferase fusionconstruct to a luciferase output level of greater than 70; and iva) at7.5 μM concentration, does not activate a negative control V9m cell lineexpressing TK-renilla luciferase reporter to a level greater than 20;and va) inhibits the enzymatic activity of recombinant 15-PGDH proteinat an IC₅₀ of less than 1 μM

In other embodiments, the 15-PGDH inhibitor can ib) at 2.5 μMconcentration, stimulate a Vaco503 reporter cell line expressing a15-PGDH luciferase fusion construct to increase luciferase output; iib)at 2.5 μM concentration stimulate a V9m reporter cell line expressing a15-PGDH luciferase fusion construct to increase luciferase output; iiib)at 7.5 μM concentration stimulate a LS174T reporter cell line expressinga 15-PGDH luciferase fusion construct to increase luciferase output;ivb) at 7.5 μM concentration, does not activate a negative control V9mcell line expressing TK-renilla luciferase reporter to a luciferaselevel greater than 20% above background; and vb) inhibits the enzymaticactivity of recombinant 15-PGDH protein at an IC₅₀ of less than 1 μM.

In other embodiments, the 15-PGDH inhibitor can inhibit the enzymaticactivity of recombinant 15-PGDH at an IC₅₀ of less than 1 μM, orpreferably at an IC₅₀ of less than 250 nM, or more preferably at an IC₅₀of less than 50 nM, or more preferably at an IC₅₀ of less than 10 nM, ormore preferably at an IC₅₀ of less than 5 nM at a recombinant 15-PGDHconcentration of about 5 nM to about 10 nM.

In other embodiments, the 15-PGDH inhibitor can increase the cellularlevels of PGE-2 following stimulation of an A459 cell with anappropriate agent, for example IL1-beta.

In some embodiments, a 15-PGDH inhibitor having formula (V) can includea compound with the following formula (XIII):

and pharmaceutically acceptable salts thereof.

Advantageously, the 15-PGDH inhibitor having formula (XIII) was foundto: i) inhibit recombinant 15-PGDH at 3 nM concentration when enzymeconcentration was approximately 6 nM; ii) increase PGE₂ production bycell lines with an EC50 of around 20 nM; iii) is chemically stable inaqueous solutions over broad pH range; iv) is chemically stable whenincubated with mouse, rat and human liver extracts, v) shows 33 minutesplasma half-life when injected IP into mice; viii) shows no immediatetoxicity over 24 hours when injected IP into mice at 50 mg/kg bodyweight, and ix) is soluble in water (pH=3) at 1 mg/mL; ix) elevates PGE2levels in the colon, lung, liver and bone marrow following an IP dose of10 mg/kg body weight; x) increases homing of hematopoietic stem cellsfollowing bone marrow transplantation to a mouse when administered at 10mg/kg body weight.

In other embodiments, a 15-PGDH inhibitor having formula (XIII) caninclude a compound with the following formula (XIIIa):

-   -   and pharmaceutically acceptable salts thereof.

In still other embodiments, a 15-PGDH inhibitor having formula (XIII)can include a compound with the following formula (XIIIb):

-   -   and pharmaceutically acceptable salts thereof.

In other embodiments, the 15-PDHG inhibitor can comprise a (+) or (−)optical isomer of a 15-PGDH inhibitor having formula (XIII). In stillother embodiments, the 15-PDHG inhibitor can comprise a mixture at leastone of a (+) or (−) optical isomer of a 15-PGDH inhibitor having formula(XIII). For example, the 15-PGDH inhibitor can comprise a mixture of:less than about 50% by weight of the (−) optical isomer of a 15-PGDHinhibitor having formula (XIII) and greater than about 50% by weight ofthe (+) optical isomer of a 15-PGDH inhibitor having formula (XIII),less than about 25% by weight of the (−) optical isomer of a 15-PGDHinhibitor having formula (XIII) and greater than about 75% by weight ofthe (+) optical isomer of a 15-PGDH inhibitor having formula (XIII),less than about 10% by weight of the (−) optical isomer of a 15-PGDHinhibitor having formula (XIII) and greater than about 90% by weight ofthe (+) optical isomer of a 15-PGDH inhibitor having formula (XIII),less than about 1% by weight of the (−) optical isomer of a 15-PGDHinhibitor having formula (XIII) and greater than about 99% by weight ofthe (+) optical isomer of a 15-PGDH inhibitor having formula (XIII),greater than about 50% by weight of the (−) optical isomer of a 15-PGDHinhibitor having formula (XIII) and less than about 50% by weight of the(+) optical isomer of a 15-PGDH inhibitor having formula (XIII), greaterthan about 75% by weight of the (−) optical isomer of a 15-PGDHinhibitor having formula (XIII) and less than about 25% by weight of the(+) optical isomer of a 15-PGDH inhibitor having formula (XIII), greaterthan about 90% by weight of the (−) optical isomer of a 15-PGDHinhibitor having formula (XIII) and less than about 10% by weight of the(+) optical isomer of a 15-PGDH inhibitor having formula (XIII), orgreater than about 99% by weight of the (−) optical isomer of a 15-PGDHinhibitor having formula (XIII) and less than about 1% by weight of the(+) optical isomer of a 15-PGDH inhibitor having formula (XIII).

In a still further embodiment, the 15-PGDH inhibitor can consistessentially of or consist of the (+) optical isomer of a 15-PGDHinhibitor having formula (XIII). In yet another embodiment, the PGDHinhibitor can consist essentially of or consist of the (−) opticalisomer of a 15-PGDH inhibitor having formula (XIII).

The 15-PGDH inhibitors described herein can be used for the preventionor the treatment of diseases that are associated with 15-PGDH and/ordecreased prostaglandin levels and/or where it desirable to increaseprostaglandin levels in the subject. For example, as discussed above, itis known that prostaglandins play an important role in hair growth.Specifically, internal storage of various types (A₂, F_(2a), E₂) ofprostaglandins in the various compartments of hair follicles or theiradjacent skin environments has been shown to be essential in maintainingand increasing hair density (Colombe L et. al, 2007, Exp. Dermatol,16(9), 762-9). It has been reported that 15-PGDH, which is involved inthe degradation of prostaglandins is present in the hair follicle dermalpapillae, inactivates prostaglandins, especially, PGF_(2a) and PGE₂, tocause scalp damage and alopecia (Michelet J F et. al., 2008, Exp.Dermatol, 17(10), 821-8). Thus, the compounds described herein, whichhave a suppressive or inhibitory activity against 15-PGDH that degradesprostaglandins, can improve scalp damage, prevent alopecia and promotehair growth and be used in a pharmaceutical composition for theprevention of alopecia and the promotion of hair growth.

In other embodiments, the 15-PGDH inhibitors described herein can beused in a pharmaceutical composition for promoting and/or inducingand/or stimulating pigmentation of the skin and/or skin appendages,and/or as an agent for preventing and/or limiting depigmentation and/orwhitening of the skin and/or skin appendages, in particular as an agentfor preventing and/or limiting canities.

In some embodiments, the 15-PGDH inhibitor can be applied to skin of asubject, e.g., in a topical application, to promote and/or stimulatepigmentation of the skin and/or hair growth, inhibit hair loss, and/ortreat skin damage or inflammation, such as skin damage caused byphysical or chemical irritants and/or UV-exposure.

In still other embodiments, the 15-PGDH inhibitors described herein canbe used in a pharmaceutical composition for the prevention or thetreatment of cardiovascular disease and/or diseases of vascularinsufficiency, such as Raynaud's disease, Buerger's disease, diabeticneuropathy, and pulmonary artery hypertension. Prostaglandins includingprostaglandin homologues produced in the body have been known tomaintain the proper action of the blood vessel wall, especially tocontribute to vasodilation for blood flow, preventing plateletaggregation and modulating the proliferation of smooth muscle thatsurrounds blood vessel walls (Yan. Cheng et. al., 2006, J. Clin.,Invest). In addition, the inhibition of prostaglandins production or theloss of their activity causes the degeneration of the endothelium in theblood vessel walls, platelet aggregation and the dysfunction of cellularmechanism in the smooth muscle. Among others, the production ofprostaglandins in blood vessels was shown to be decreased inhypertension patients, including pulmonary artery hypertension.

In other embodiments, the 15-PGDH inhibitors described herein can beused in a pharmaceutical composition for the prevention or the treatmentof oral, intestinal, and/or gastrointestinal injury or diseases, orinflammatory bowel disease, such as oral ulcers, gum disease, gastritis,colitis, ulcerative colitis, and gastric ulcers. Gastritis and gastriculcer, representatives of the gastrointestinal diseases, are defined asthe conditions where gastrointestinal mucus membrane is digested bygastric acid to form ulcer. In the stomach walls generally consisting ofmucosa, submucosa, muscle layer and serosa, gastric ulcer even damagessubmucosa and muscle layer, while gastritis damages mucosa only.Although the morbidity rates of gastritis and gastric ulcer arerelatively high, the causes thereof have not been clarified yet. Untilnow, they are known to be caused by an imbalance between aggressivefactors and defensive factors, that is, the increase in aggressivefactors such as the increase in gastric acid or pepsin secretion, or thedecrease in defensive factors such as structural or morphologicaldeficit of the gastric mucus membrane, the decrease in mucus andbicarbonate ion secretion, the decrease in prostaglandin production, orthe like.

Currently available therapeutic agents for gastritis and gastric ulcercomprise various drugs for strengthening the defensive factors such asan antacid, which does not affect, gastric acid secretion butneutralizes gastric acid that has been already produced, an inhibitor ofgastric acid secretion, a promoter of prostaglandin secretion, and acoating agent for stomach walls. Especially, prostaglandins are known tobe essential in maintaining the mechanism for protecting and defendinggastric mucus membrane (Wallace J L., 2008, Physiol Rev., 88(4),1547-65, S. J. Konturek et al., 2005, Journal of Physiology andPharmacology, 56(5)). In view of the above, since the 15-PGDH inhibitorsdescribed herein show a suppressive or inhibitory activity against15-PGDH, which degrades prostaglandins that protect gastric mucusmembrane, they can be effective for the prevention or the treatment ofgastrointestinal diseases, inter alia, gastritis and gastric ulcer.

Moreover, 15-PGDH inhibitors would also be expected to protect fromother form of intestinal injury that would include toxicity fromradiation, toxicity from chemotherapy, and chemotherapy inducedmucositis.

In the kidney, prostaglandins modulate renal blood flow and may serve toregulate urine formation by both renovascular and tubular effects. Inclinical studies, PGE₁ has been used to improve creatinine clearance inpatients with chronic renal disease, to prevent graft rejection andcyclosporine toxicity in renal transplant patients, to reduce theurinary albumin excretion rate and N-acetyl-beta-D-glucosaminidaselevels in patients with diabetic nephropathy (see Porter, Am., 1989, J.Cardiol., 64: 22E-26E). In addition, U.S. Pat. No. 5,807,895 discloses amethod of preventing renal dysfunction by intravenous administration ofprostaglandins such as PGE₁, PGE₂ and PGI₂. Furthermore, it has beenreported that prostaglandins serve as vasodilators in the kidney, and,thus, the inhibition of prostaglandin production in the kidney resultsin renal dysfunction (Hao. C M, 2008, Annu Rev Physiol, 70,357.about.77).

Thus, the 15-PGDH inhibitors described herein, which have a suppressiveor inhibitory activity against 15-PGDH that degrades prostaglandins, maybe effective in the prevention or the treatment of renal diseases thatare associated with renal dysfunction.

The term “renal dysfunction” as used herein includes such manifestationsas follows: lower than normal creatinine clearance, lower than normalfree water clearance, higher than normal blood urea, nitrogen, potassiumand/or creatinine levels, altered activity of kidney enzymes such asgamma glutamyl synthetase, alanine phosphatidase,N-acetyl-beta-D-glucosaminidase, or beta-w-microglobulin; and increaseover normal levels of macroalbuminuria.

Prostaglandins including PGE₁, PGE₂ and PGF_(2a) have also been shown tostimulate bone resorption and bone formation to increase the volume andthe strength of the bone (H. Kawaguchi et. al., Clinical Orthop. Rel.Res., 313, 1995; J. Keller et al., Eur. Jr. Exp. Musculoskeletal Res.,1, 1992, 8692). Considering that 15-PGDH inhibits the activities ofprostaglandins as mentioned in the above, the inhibition of 15-PGDHactivity may lead to the promotion of bone resorption and bone formationthat are inhibited by 15-PGDH. Thus, the 15-PGDH inhibitors describedherein can be effective for the promotion of bone resorption and boneformation by inhibiting 15-PGDH activity. 15-PGDH inhibitors can also beused to increase bone density, treat osteoporosis, promote healing offractures, or promote healing after bone surgery or joint replacement,or to promote healing of bone to bone implants, bone to artificialimplants, dental implants, and bone grafts.

In yet other embodiments, the 15-PGDH inhibitors described herein caneffective for treating 15-PGDH expressing cancers. Inhibition of 15-PGDHcan inhibit the growth, proliferation, and metastasis of 15-PGDHexpressing cancers.

In still other embodiments, the 15-PGDH inhibitors described herein canbe effective for wound healing. Among various prostaglandins, PGE₂ isknown to serve as a mediator for wound healing. Therefore, when skin isinjured by wounds or burns, the inhibition of 15-PGDH activity canproduce the treatment effect of the wounds or the burns by PGE₂.

Additionally, as discussed above, increased prostaglandin levels havebeen shown to stimulate signaling through the Wnt signaling pathway viaincreased beta-catenin mediated transcriptional activity. Wnt signalingis known to be a key pathway employed by tissue stem cells. Hence,15-PGDH inhibitors described herein may be utilized to increase tissuestem cell numbers for purposes that would include promoting tissueregeneration or repair in organs that would include liver, colon, andbone marrow. In addition, 15-PGDH inhibitors described herein may beutilized to promote tissue regeneration or repair in additional organsthat would include but are not limited to brain, eye, cornea, retina,lung, heart, stomach, small intestine, pancreas, beta-cells of thepancreas, kidney, bone, cartilage, peripheral nerve.

Syndromic conditions, traumatic injuries, chronic conditions, medicalinterventions, or other conditions that cause or are associated withtissue damage and a need for tissue repair, and thus, suitable fortreatment or amelioration using the methods described herein, include,but are not limited to, acute coronary syndrome, acute lung injury(ALI), acute myocardial infarction (AMI), acute respiratory distresssyndrome (ARDS), arterial occlusive disease, arteriosclerosis, articularcartilage defect, aseptic systemic inflammation, atheroscleroticcardiovascular disease, autoimmune disease, bone fracture, bonefracture, brain edema, brain hypoperfusion, Buerger's disease, burns,cancer, cardiovascular disease, cartilage damage, cerebral infarct,cerebral ischemia, cerebral stroke, cerebrovascular disease,chemotherapy-induced neuropathy, chronic infection, chronic mesentericischemia, claudication, congestive heart failure, connective tissuedamage, contusion, coronary artery disease (CAD), critical limb ischemia(CLI), Crohn's disease, deep vein thrombosis, deep wound, delayed ulcerhealing, delayed wound-healing, diabetes (type I and type II), diabetes,diabetic neuropathy, diabetes induced ischemia, disseminatedintravascular coagulation (DIC), embolic brain ischemia,graft-versus-host disease, frostbite, hereditary hemorrhagictelengiectasiaischemic vascular disease, hyperoxic injury, hypoxia,inflammation, inflammatory bowel disease, inflammatory disease, injuredtendons, intermittent claudication, intestinal ischemia, ischemia,ischemic brain disease, ischemic heart disease, ischemic peripheralvascular disease, ischemic placenta, ischemic renal disease, ischemicvascular disease, ischemic-reperfusion injury, laceration, left maincoronary artery disease, limb ischemia, lower extremity ischemia,myocardial infarction, myocardial ischemia, organ ischemia,osteoarthritis, osteoporosis, osteosarcoma, Parkinson's disease,peripheral arterial disease (PAD), peripheral artery disease, peripheralischemia, peripheral neuropathy, peripheral vascular disease,pre-cancer, pulmonary edema, pulmonary embolism, remodeling disorder,renal ischemia, retinal ischemia, retinopathy, sepsis, skin ulcers,solid organ transplantation, spinal cord injury, stroke,subchondral-bone cyst, thrombosis, thrombotic brain ischemia, tissueischemia, transient ischemic attack (TIA), traumatic brain injury,ulcerative colitis, vascular disease of the kidney, vascularinflammatory conditions, von Hippel-Lindau syndrome, and wounds totissues or organs.

Other illustrative examples of genetic disorders, syndromic conditions,traumatic injuries, chronic conditions, medical interventions, or otherconditions that cause or are associated with tissue damage and a needfor tissue repair suitable for treatment or amelioration using themethods of the present invention, include, ischemia resulting fromsurgery, chemotherapy, radiation therapy, or cell, tissue, or organtransplant or graft.

In various embodiments, the methods of the invention are suitable fortreating cerebrovascular ischemia, myocardial ischemia, limb ischemia(CLI), myocardial ischemia (especially chronic myocardial ischemia),ischemic cardiomyopathy, cerebrovascular ischemia, renal ischemia,pulmonary ischemia, intestinal ischemia, and the like.

In some embodiments, the ischemia is associated with at least one ofacute coronary syndrome, acute lung injury (ALI), acute myocardialinfarction (AMI), acute respiratory distress syndrome (ARDS), arterialocclusive disease, arteriosclerosis, articular cartilage defect, asepticsystemic inflammation, atherosclerotic cardiovascular disease,autoimmune disease, bone fracture, bone fracture, brain edema, brainhypoperfusion, Buerger's disease, burns, cancer, cardiovascular disease,cartilage damage, cerebral infarct, cerebral ischemia, cerebral stroke,cerebrovascular disease, chemotherapy-induced neuropathy, chronicinfection, chronic mesenteric ischemia, claudication, congestive heartfailure, connective tissue damage, contusion, coronary artery disease(CAD), critical limb ischemia (CLI), Crohn's disease, deep veinthrombosis, deep wound, delayed ulcer healing, delayed wound-healing,diabetes (type I and type II), diabetic neuropathy, diabetes inducedischemia, disseminated intravascular coagulation (DIC), embolic brainischemia, graft-versus-host disease, hereditary hemorrhagictelengiectasiaischemic vascular disease, hyperoxic injury, hypoxia,inflammation, inflammatory bowel disease, inflammatory disease, injuredtendons, intermittent claudication, intestinal ischemia, ischemia,ischemic brain disease, ischemic heart disease, ischemic peripheralvascular disease, ischemic placenta, ischemic renal disease, ischemicvascular disease, ischemic-reperfusion injury, laceration, left maincoronary artery disease, limb ischemia, lower extremity ischemia,myocardial infarction, myocardial ischemia, organ ischemia,osteoarthritis, osteoporosis, osteosarcoma, Parkinson's disease,peripheral arterial disease (PAD), peripheral artery disease, peripheralischemia, peripheral neuropathy, peripheral vascular disease,pre-cancer, pulmonary edema, pulmonary embolism, remodeling disorder,renal ischemia, retinal ischemia, retinopathy, sepsis, skin ulcers,solid organ transplantation, spinal cord injury, stroke,subchondral-bone cyst, thrombosis, thrombotic brain ischemia, tissueischemia, transient isc hemic attack (TIA), traumatic brain injury,ulcerative colitis, vascular disease of the kidney, vascularinflammatory conditions, von Hippel-Lindau syndrome, and wounds totissues or organs.

In some embodiments, the 15-PGDH inhibitor can be administered to apreparation of hematopoietic stem cells, such as peripheral bloodhematopoietic stem cells or umbilical cord stem cells of the subject, toincrease the fitness of the stem cell preparation as a donor graft or todecrease the number of units of umbilical cord blood required fortransplantation.

Hematopoietic stem cells are multipotent stem cells that give rise toall the blood cell types of an organism, including myeloid (e.g.,monocytes and macrophages, neutrophils, basophils, eosinophils,erythrocytes, megakaryocytes/platelets, dendritic cells), and lymphoidlineages (e.g., T-cells, B-cells, NK-cells), and others known in the art(See Fei, R., et al, U.S. Pat. No. 5,635,387; McGlave, et al, U.S. Pat.No. 5,460,964; Simmons, P., et al, U.S. Pat. No. 5,677,136; Tsukamoto,et al, U.S. Pat. No. 5,750,397; Schwartz, et al, U.S. Pat. No.5,759,793; DiGuisto, et al, U.S. Pat. No. 5,681,599; Tsukamoto, et al,U.S. Pat. No. 5,716,827). Hematopoietic stem cells (HSCs) give rise tocommitted hematopoietic progenitor cells (HPCs) that are capable ofgenerating the entire repertoire of mature blood cells over the lifetimeof an organism.

Hematopoietic stem cells and hematopoietic progenitor cells aredescribed herein generally as hematopoietic stem cells unless notedotherwise and can refer to cells or populations identified by thepresence of the antigenic marker CD34 (CD34⁺). In some embodiments, thehematopoietic stem cells can be identified by the presence of theantigenic marker CD34 and the absence of lineage (lin) markers and aretherefore characterized as CD34⁺/lin⁻ cells.

The hematopoietic stem cells used in the methods described herein may beobtained from any suitable source of hematopoietic stem and progenitorcells and can be provided as a high purified population of hematopoieticstem cells or as composition that includes about 0.01% to about 100% ofhematopoietic stem cells. For example, hematopoietic stem cells may beprovided in compositions, such as unfractionated bone marrow (where thehematopoietic stem cells comprise less than about 1% of the bone marrowcell population), umbilical cord blood, placental blood, placenta, fetalblood, fetal liver, fetal spleen, Wharton's jelly, or mobilizedperipheral blood.

Suitable sources of hematopoietic stem cells can be isolated or obtainedfrom an organ of the body containing cells of hematopoietic origin. Theisolated cells can include cells that are removed from their originalenvironment. For example, a cell is isolated if it is separated fromsome or all of the components that normally accompany it in its nativestate. For example, an “isolated population of cells,” an “isolatedsource of cells,” or “isolated hematopoietic stem cells” and the like,as used herein, refer to in vitro or ex vivo separation of one or morecells from their natural cellular environment, and from association withother components of the tissue or organ, i.e., it is not significantlyassociated with in vivo substances.

Hematopoietic stem cells can be obtained or isolated from bone marrow ofadults, which includes femurs, hip, ribs, sternum, and other bones. Bonemarrow aspirates containing hematopoietic stem cells can be obtained orisolated directly from the hip using a needle and syringe. Other sourcesof hematopoietic stem cells include umbilical cord blood, placentalblood, mobilized peripheral blood, Wharton's jelly, placenta, fetalblood, fetal liver, or fetal spleen. In particular embodiments,harvesting a sufficient quantity of hematopoietic stem cells for use intherapeutic applications may require mobilizing the stem and progenitorcells in the donor.

“Hematopoietic stem cell mobilization” refers to the release of stemcells from the bone marrow into the peripheral blood circulation for thepurpose of leukapheresis, prior to stem cell transplantation. Byincreasing the number of stem cells harvested from the donor, the numberof stem cells available for therapeutic applications can besignificantly improved. Hematopoietic growth factors, e.g., granulocytecolony stimulating factor (G-CSF) or chemotherapeutic agents often areused to stimulate the mobilization. Commercial stem cell mobilizationdrugs exist and can be used in combination with G-CSF to mobilizesufficient quantities of hematopoietic stem and progenitor cells fortransplantation into a subject. For example, G-CSF and Mozobil (GenzymeCorporation) can be administered to a donor in order to harvest asufficient number of hematopoietic cells for transplantation. Othermethods of mobilizing hematopoietic stem cells would be apparent to onehaving skill in the art.

In some embodiments, hematopoietic stem and progenitor cells (HSPCs) areobtained from umbilical cord blood. Cord blood can be harvestedaccording to techniques known in the art {see, e.g., U.S. Pat. Nos.7,147,626 and 7,131,958, herein incorporated by reference for suchmethodologies).

In one embodiment, HSPCs can be obtained from pluripotent stem cellsources, e.g., induced pluripotent stem cells (iPSCs) and embryonic stemcells (ESCs). As used herein, the term “induced pluripotent stem cell”or “iPSC” refers to a non-pluripotent cell that has been reprogrammed toa pluripotent state. Once the cells of a subject have been reprogrammedto a pluripotent state, the cells can then be programmed to a desiredcell type, such as a hematopoietic stem or progenitor cell. As usedherein, the term “reprogramming” refers to a method of increasing thepotency of a cell to a less differentiated state. As used herein, theterm “programming” refers to a method of decreasing the potency of acell or differentiating the cell to a more differentiated state.

In some embodiments, the hematopoietic stem cells can be administered orcontacted ex vivo with one or more 15-PGDH inhibitors described hereinto provide a therapeutic composition. In one embodiment, the therapeuticcompositions of the can include a population of hematopoietic stem cellstreated ex vivo with a one or more 15-PGDH inhibitor. In certainembodiments, the therapeutic composition comprising the enhanced HSPCsis whole bone marrow, umbilical cord blood, or mobilized peripheralblood.

In particular embodiments, the therapeutic composition includes apopulation of cells, wherein the population of cells is about 95% toabout 100% hematopoietic stem cells. The invention contemplates, inpart, that using therapeutic compositions of highly purifiedhematopoietic stem cells, e.g., a composition comprising a population ofcells wherein the cells comprise about 95% hematopoietic stem cells, mayimprove the efficiency of stem cell therapies. Currently practicedmethods of transplantations typically use unfractionated mixtures ofcells where hematopoietic stem cells comprise less than 1% of the totalcell population.

In some embodiments, the therapeutic composition comprises a populationof cells, wherein the population of cells comprises less than about0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, or 30% hematopoietic stemcells. The population of cells in some embodiments comprises less thanabout 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, or 30% hematopoieticstem cells. In other embodiments, the population of cells is about 0.1%to about 1%, about 1% to about 3%, about 3% to about 5%, about 10%-15%,about 15%-20%, about 20%-25%, about 25%-30%, about 30%-35%, about35%-40%, about 40%-45%, about 45%-50%, about 60%-70%, about 70%-80%,about 80%-90%, about 90%-95%, or about 95% to about 100% hematopoieticstem cells.

Hematopoietic stem cells in the therapeutic compositions of theinvention can be autologous/autogenetic (“self) or non-autologous(“non-self,” e.g., allogeneic, syngeneic or xenogeneic) relative to asubject to which the therapeutic composition is to be administered.“Autologous,” as used herein, refers to cells from the same subject.“Allogeneic,” as used herein, refers to cells of the same species thatdiffer genetically to the cell in comparison. “Syngeneic,” as usedherein, refers to cells of a different subject that are geneticallyidentical to the cell in comparison. “Xenogeneic,” as used herein,refers to cells of a different species to the cell in comparison.

Hematopoietic stem cells for use in the methods of the present inventionmay be depleted of mature hematopoietic cells such as T cells, B cells,NK cells, dendritic cells, monocytes, granulocytes, erythroid cells, andtheir committed precursors from bone marrow aspirate, umbilical cordblood, or mobilized peripheral blood (mobilized leukapheresis product).Mature, lineage committed cells are depleted by immunodepletion, forexample, by labeling solid substrates with antibodies that bind to apanel of so-called “lineage” antigens: CD2, CD3, CD11b, CD14, CD15,CD16, CD79, CD56, CD123, and CD235a. A subsequent step can be performedto further purify the population of cells, in which a substrate labeledwith antibodies that bind to the CD34⁺ antigen are used to isolateprimitive hematopoietic stem cells. Kits are commercially available forpurifying stem and progenitor cells from various cell sources and inparticular embodiments, these kits are suitable for use with the methodsdescribed herein.

In one embodiment, the amount of hematopoietic stem cells in thetherapeutic composition is at least 0.1×10⁵ cells, at least 0.5×10⁵cells, at least 1×10⁵ cells, at least 5×10⁵ cells, at least 10×10⁵cells, at least 0.5×10⁶ cells, at least 0.75×10⁶ cells, at least 1×10⁶cells, at least 1.25×10⁶ cells, at least 1.5×10⁶ cells, at least1.75×10⁶ cells, at least 2×10⁶ cells, at least 2.5×10⁶ cells, at least3×10⁶ cells, at least 4×10⁶ cells, at least 5×10⁶ cells, at least 10×10⁶cells, at least 15×10⁶ cells, at least 20×10⁶ cells, at least 25×10⁶cells, or at least 30×10⁶ cells.

In one embodiment, the amount of hematopoietic stem cells in thetherapeutic composition is the amount of HSPCs in a partial or singlecord of blood, or is at least 0.1×10⁵ cells/kg of bodyweight, at least0.5×10⁵ cells/kg of bodyweight, at least 1×10⁵ cells/kg of bodyweight,at least 5×10⁵ cells/kg of bodyweight, at least 10×10⁵ cells/kg ofbodyweight, at least 0.5×10⁶ cells/kg of bodyweight, at least 0.75×10⁶cells/kg of bodyweight, at least 1×10⁶ cells/kg of bodyweight, at least1.25×10⁶ cells/kg of bodyweight, at least 1.5×10⁶ cells/kg ofbodyweight, at least 1.75×10⁶ cells/kg of bodyweight, at least 2×10⁶cells/kg of bodyweight, at least 2.5×10⁶ cells/kg of bodyweight, atleast 3×10⁶ cells/kg of bodyweight, at least 4×10⁶ cells/kg ofbodyweight, at least 5×10⁶ cells/kg of bodyweight, at least 10×10⁶cells/kg of bodyweight, at least 15×10⁶ cells/kg of bodyweight, at least20×10⁶ cells/kg of bodyweight, at least 25×10⁶ cells/kg of bodyweight,or at least 30×10⁶ cells/kg of bodyweight.

Preparations of hematopoietic stem cells administered one or more15-PGDH inhibitors and/or therapeutic compositions that includehematopoietic stem cells and one or more 15-PGDH inhibitor can be usedfor improving hematopoietic stem cell transplants and in treatingischemia or ischemia-damaged tissue, and in reducing further damage toischemic tissue and/or repairing damage to ischemic tissue through cellrecruitment, improving vascularization in ischemic tissue, improvingtissue regeneration at sites of ischemia, decreasing ischemic tissuenecrosis or apoptosis, and/or increasing cell survival at sites ofischemia. In particular embodiments, the preparations of 15-PGDHinhibitor treated hematopoietic stem cells and/or therapeuticcompositions of 15-PGDH inhibitors and hematopoietic stem cells areuseful to subjects in need of hematopoietic reconstitution, such assubjects that have undergone or are scheduled to undergo myeloablativetherapy.

Subjects, which can be treated with the preparations of 15-PGDHinhibitor treated hematopoietic stem cells and/or therapeuticcompositions of 15-PGDH inhibitors and hematopoietic stem cells, caninclude subjects that have or that have been diagnosed with varioustypes of leukemias, anemias, lymphomas, myelomas, immune deficiencydisorders, and solid tumors. A subject also includes a human who is acandidate for stem cell transplant or bone marrow transplantation, suchas during the course of treatment for a malignant disease or a componentof gene therapy. Subjects may also include individuals or animals thatdonate stem cells or bone marrow for allogeneic transplantation. Incertain embodiments, a subject may have undergone myeloablativeirradiation therapy or chemotherapy, or may have experienced an acuteradiation or chemical insult resulting in myeloablation. In certainembodiments, a subject may have undergone irradiation therapy orchemotherapy, such as during various cancer treatments. Typical subjectsinclude animals that exhibit aberrant amounts (lower or higher amountsthan a “normal” or “healthy” subject) of one or more physiologicalactivities that can be modulated by an agent or a stem cell or marrowtransplant.

Subjects, which can be treated with the preparations of 15-PGDHinhibitor treated hematopoietic stem cells and/or therapeuticcompositions of 15-PGDH inhibitors and hematopoietic stem cells, canalso include subjects undergoing chemotherapy or radiation therapy forcancer, as well as subjects suffering from (e.g., afflicted with) nonmalignant blood disorders, particularly immunodeficiencies (e.g. SCID,Fanconi's anemia, severe aplastic anemia, or congenitalhemoglobinopathies, or metabolic storage diseases, such as Hurler'sdisease, Hunter's disease, mannosidosis, among others) or cancer,particularly hematological malignancies, such as acute leukemia, chronicleukemia (myeloid or lymphoid), lymphoma (Hodgkin's or non-Hodgkin's),multiple myeloma, myelodysplastic syndrome, or non-hematological cancerssuch as solid tumors (including breast cancer, ovarian cancer, braincancer, prostate cancer, lung cancer, colon cancer, skin cancer, livercancer, or pancreatic cancer).

Subjects may also include subjects suffering from aplastic anemia, animmune disorder (severe combined immune deficiency syndrome or lupus),myelodysplasia, thalassemia, sickle-cell disease or Wiskott-Aldrichsyndrome. In some embodiments, the subject suffers from a disorder thatis the result of an undesired side effect or complication of anotherprimary treatment, such as radiation therapy, chemotherapy, or treatmentwith a bone marrow suppressive drug, such as zidovudine, chloramphenicalor ganciclovir. Such disorders include neutropenias, anemias,thrombocytopenia, and immune dysfunction. Other subjects may havedisorders caused by an infection (e.g., viral infection, bacterialinfection or fungal infection) which causes damage to stem or progenitorcells of the bone marrow.

In addition, subjects suffering from the following conditions can alsobenefit from treatment using the preparations of 15-PGDH inhibitortreated hematopoietic stem cells and/or therapeutic compositions of15-PGDH inhibitors and hematopoietic stem cells: lymphocytopenia,lymphorrhea, lymphostasis, erythrocytopenia, erthrodegenerativedisorders, erythroblastopenia, leukoerythroblastosis; erythroclasis,thalassemia, myelodysplasia, myelofibrosis, thrombocytopenia,disseminated intravascular coagulation (DIC), immune (autoimmune)thrombocytopenic purpura (ITP), HIV inducted ITP, myelodysplasia;thrombocytotic disease, thrombocytosis, congenital neutropenias (such asKostmann's syndrome and Schwachman-Diamond syndrome), neoplasticassociated neutropenias, childhood and adult cyclic neutropaenia;post-infective neutropaenia; myelodysplastic syndrome; neutropaeniaassociated with chemotherapy and radiotherapy; chronic granulomatousdisease; mucopolysaccharidoses; Diamond Blackfan Anemia; Sickle celldisease; or Beta thalassemia major.

In other embodiments, the preparations of 15-PGDH inhibitor treatedhematopoietic stem cells and/or therapeutic compositions or 15-PGDHinhibitors and hematopoietic stem cells can be used in cell-basedtherapy for treating ischemic tissue or treating or ameliorating one ormore symptoms associated with tissue ischemia, including, but notlimited to, impaired, or loss of, organ function (including withoutlimitation impairments or loss of brain, kidney, or heart function),cramping, claudication, numbness, tingling, weakness, pain, reducedwound healing, inflammation, skin discoloration, and gangrene.

In one embodiment, the subject exhibits at least one symptom of anischemic tissue or tissue damaged by ischemia. In particularembodiments, the subject is a human who is has or who is at risk ofhaving an ischemic tissue or tissue damaged by ischemia, e.g., a subjectthat has diabetes, peripheral vascular disease, thromboangiitisobliterans, vasculitis, cardiovascular disease, coronary artery diseaseor heart failure, or cerebrovascular disease, cardiovascular disease, orcerebrovascular disease.

Illustrative examples of genetic disorders, syndromic conditions,traumatic injuries, chronic conditions, medical interventions, or otherconditions that cause or are associated with ischemia, or increase therisk of ischemia in a subject, or cause a subject to exhibit more ormore symptoms of ischemia, and thus, suitable for treatment oramelioration using the methods described herein, include, but are notlimited to, acute coronary syndrome, acute lung injury (ALI), acutemyocardial infarction (AMI), acute respiratory distress syndrome (ARDS),arterial occlusive disease, arteriosclerosis, articular cartilagedefect, aseptic systemic inflammation, atherosclerotic cardiovasculardisease, autoimmune disease, bone fracture, bone fracture, brain edema,brain hypoperfusion, Buerger's disease, burns, cancer, cardiovasculardisease, cartilage damage, cerebral infarct, cerebral ischemia, cerebralstroke, cerebrovascular disease, chemotherapy-induced neuropathy,chronic infection, chronic mesenteric ischemia, claudication, congestiveheart failure, connective tissue damage, contusion, coronary arterydisease (CAD), critical limb ischemia (CLI), Crohn's disease, deep veinthrombosis, deep wound, delayed ulcer healing, delayed wound-healing,diabetes (type I and type II), diabetic neuropathy, diabetes inducedischemia, disseminated intravascular coagulation (DIC), embolic brainischemia, graft-versus-host disease, frostbite, hereditary hemorrhagictelengiectasiaischemic vascular disease, hyperoxic injury, hypoxia,inflammation, inflammatory bowel disease, inflammatory disease, injuredtendons, intermittent claudication, intestinal ischemia, ischemia,ischemic brain disease, ischemic heart disease, ischemic peripheralvascular disease, ischemic placenta, ischemic renal disease, ischemicvascular disease, ischemic-reperfusion injury, laceration, left maincoronary artery disease, limb ischemia, lower extremity ischemia,myocardial infarction, myocardial ischemia, organ ischemia,osteoarthritis, osteoporosis, osteosarcoma, Parkinson's disease,peripheral arterial disease (PAD), peripheral artery disease, peripheralischemia, peripheral neuropathy, peripheral vascular disease,pre-cancer, pulmonary edema, pulmonary embolism, remodeling disorder,renal ischemia, retinal ischemia, retinopathy, sepsis, skin ulcers,solid organ transplantation, spinal cord injury, stroke,subchondral-bone cyst, thrombosis, thrombotic brain ischemia, tissueischemia, transient ischemic attack (TIA), traumatic brain injury,ulcerative colitis, vascular disease of the kidney, vascularinflammatory conditions, von Hippel-Lindau syndrome, and wounds totissues or organs.

Other illustrative examples of genetic disorders, syndromic conditions,traumatic injuries, chronic conditions, medical interventions, or otherconditions that cause or are associated with ischemia, or increase therisk of ischemia in a subject, or cause a subject to exhibit more ormore symptoms of ischemia suitable for treatment or amelioration usingthe methods of the present invention, include, ischemia resulting fromsurgery, chemotherapy, radiation therapy, or cell, tissue, or organtransplant or graft.

In various embodiments, the methods of the invention are suitable fortreating cerebrovascular ischemia, myocardial ischemia, limb ischemia(CLI), myocardial ischemia (especially chronic myocardial ischemia),ischemic cardiomyopathy, cerebrovascular ischemia, renal ischemia,pulmonary ischemia, intestinal ischemia, and the like.

In various embodiments, the invention contemplates that the therapeuticcell compositions disclosed herein can be used to treat an ischemictissue in which it is desirable to increase the blood flow, oxygensupply, glucose supply, or supply of nutrients to the tissue.

In some embodiments, the 15-PGDH inhibitor can be administered to apreparation of tissue stem cells, such as neural stem stems, mesenchymalstem cells, or stem cells that can generate other tissues, and/or apreparation of pluripotent stem cells.

In one embodiment, tissue stems cells can be obtained from pluripotentstem cell sources, e.g., induced pluripotent stem cells (iPSCs) andembryonic stem cells (ESCs). As used herein, the term “inducedpluripotent stem cell” or “iPSC” refers to a non-pluripotent cell thathas been reprogrammed to a pluripotent state. Once the cells of asubject have been reprogrammed to a pluripotent state, the cells canthen be programmed to a desired cell type, such as a hematopoietic stemor progenitor cell. As used herein, the term “reprogramming” refers to amethod of increasing the potency of a cell to a less differentiatedstate. As used herein, the term “programming” refers to a method ofdecreasing the potency of a cell or differentiating the cell to a moredifferentiated state.

In some embodiments, the tissue stem cells and/or pluripotent stem cellscan be administered or contacted ex vivo with one or more 15-PGDHinhibitors described herein to provide a therapeutic composition. In oneembodiment, the therapeutic compositions of the can include a populationof tissue stem cells treated ex vivo with a one or more 15-PGDHinhibitor.

In particular embodiments, the therapeutic composition includes apopulation of cells, wherein the population of cells is about 95% toabout 100% tissue stem cells. The invention contemplates, in part, thatusing therapeutic compositions of highly purified tissue stem cells,e.g., a composition comprising a population of cells wherein the cellscomprise about 95% tissue stem cells, may improve the efficiency of stemcell therapies

In some embodiments, the therapeutic composition comprises a populationof cells, wherein the population of cells comprises less than about0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, or 30% tissue stem cells.The population of cells in some embodiments comprises less than about0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, or 30% tissue stem cells. Inother embodiments, the population of cells is about 0.1% to about 1%,about 1% to about 3%, about 3% to about 5%, about 10%-15%, about15%-20%, about 20%-25%, about 25%-30%, about 30%-35%, about 35%-40%,about 40%-45%, about 45%-50%, about 60%-70%, about 70%-80%, about80%-90%, about 90%-95%, or about 95% to about 100% tissue stem cells.

Tissue stem cells in the therapeutic compositions of the invention canbe autologous/autogeneic (“self) or non-autologous (“non-self,” e.g.,allogeneic, syngeneic or xenogeneic) relative to a subject to which thetherapeutic composition is to be administered. “Autologous,” as usedherein, refers to cells from the same subject. “Allogeneic,” as usedherein, refers to cells of the same species that differ genetically tothe cell in comparison. “Syngeneic,” as used herein, refers to cells ofa different subject that are genetically identical to the cell incomparison. “Xenogeneic,” as used herein, refers to cells of a differentspecies to the cell in comparison.

Preparations of tissue stem cells administered one or more 15-PGDHinhibitors and/or therapeutic compositions that include tissue stemcells and one or more 15-PGDH inhibitor can be used for improving tissuestem cell transplants and in treating damaged tissue, and in reducingfurther tissue damage tissue and/or potentiating repair to damagedtissue through stem cell recruitment and/or increasing cell survival atsites of tissue damage.

Syndromic conditions, traumatic injuries, chronic conditions, medicalinterventions, or other conditions that cause or are associated withtissue damage and a need for tissue repair, and thus, suitable fortreatment or amelioration using the methods described herein, include,but are not limited to, acute coronary syndrome, acute lung injury(ALI), acute myocardial infarction (AMI), acute respiratory distresssyndrome (ARDS), arterial occlusive disease, arteriosclerosis, articularcartilage defect, aseptic systemic inflammation, atheroscleroticcardiovascular disease, autoimmune disease, bone fracture, bonefracture, brain edema, brain hypoperfusion, Buerger's disease, burns,cancer, cardiovascular disease, cartilage damage, cerebral infarct,cerebral ischemia, cerebral stroke, cerebrovascular disease,chemotherapy-induced neuropathy, chronic infection, chronic mesentericischemia, claudication, congestive heart failure, connective tissuedamage, contusion, coronary artery disease (CAD), critical limb ischemia(CLI), Crohn's disease, deep vein thrombosis, deep wound, delayed ulcerhealing, delayed wound-healing, diabetes (type I and type II), diabetes,diabetic neuropathy, diabetes induced ischemia, disseminatedintravascular coagulation (DIC), embolic brain ischemia,graft-versus-host disease, frostbite, hereditary hemorrhagictelengiectasiaischemic vascular disease, hyperoxic injury, hypoxia,inflammation, inflammatory bowel disease, inflammatory disease, injuredtendons, intermittent claudication, intestinal ischemia, ischemia,ischemic brain disease, ischemic heart disease, ischemic peripheralvascular disease, ischemic placenta, ischemic renal disease, ischemicvascular disease, ischemic-reperfusion injury, laceration, left maincoronary artery disease, limb ischemia, lower extremity ischemia,myocardial infarction, myocardial ischemia, organ ischemia,osteoarthritis, osteoporosis, osteosarcoma, Parkinson's disease,peripheral arterial disease (PAD), peripheral artery disease, peripheralischemia, peripheral neuropathy, peripheral vascular disease,pre-cancer, pulmonary edema, pulmonary embolism, remodeling disorder,renal ischemia, retinal ischemia, retinopathy, sepsis, skin ulcers,solid organ transplantation, spinal cord injury, stroke,subchondral-bone cyst, thrombosis, thrombotic brain ischemia, tissueischemia, transient ischemic attack (TIA), traumatic brain injury,ulcerative colitis, vascular disease of the kidney, vascularinflammatory conditions, von Hippel-Lindau syndrome, and wounds totissues or organs.

Other illustrative examples of genetic disorders, syndromic conditions,traumatic injuries, chronic conditions, medical interventions, or otherconditions that cause or are associated with tissue damage and a needfor tissue repair suitable for treatment or amelioration using themethods of the present invention, include, ischemia resulting fromsurgery, chemotherapy, radiation therapy, or cell, tissue, or organtransplant or graft.

In various embodiments, the methods of the invention are suitable fortreating cerebrovascular ischemia, myocardial ischemia, limb ischemia(CLI), myocardial ischemia (especially chronic myocardial ischemia),ischemic cardiomyopathy, cerebrovascular ischemia, renal ischemia,pulmonary ischemia, intestinal ischemia, and the like.

In other embodiments, the 15-PGDH inhibitor can be administered to abone marrow graft donor or a hematopoietic stem cell donor to increasethe fitness of a donor bone marrow graft or a donor hematopoietic stemcell graft.

In other embodiments, the 15-PGDH inhibitor can also be administered tobone marrow of a subject to increase stem cells in the subject or toincrease the fitness of the marrow as a donor graft.

In yet other embodiments, the 15-PGDH inhibitor can be administered to asubject to mitigate bone marrow graft rejection, to enhance bone marrowgraft engraftment, to enhance engraftment of a hematopoietic stem cellgraft, or an umbilical cord blood stem cell graft, to enhanceengraftment of a hematopoietic stem cell graft, or an umbilical cordstem cell graft, and/or to decrease the number of units of umbilicalcord blood required for transplantation into the subject. Theadministration can be, for example, following treatment of the subjector the marrow of the subject with radiation therapy, chemotherapy, orimmunosuppressive therapy.

In other embodiments, the 15-PGDH inhibitor can be administered to arecipient of a bone marrow transplant, of a hematopoietic stem celltransplant, or of an umbilical cord blood stem cell transplant, in orderto decrease the administration of other treatments or growth factors.

In some embodiments, the 15-PGDH inhibitor can be administered to asubject to enhance recovery of neutrophils following bone marrowtransplantation, following umbilical cord blood transplantation,following transplantation with hematopoietic stem cells, followingconventional chemotherapy, following radiation treatment, and inindividuals with neutropenias from diseases that include but are notlimited to aplastic anemia, myelodysplasia, myelofibrosis, neutropeniasfrom other bone marrow diseases, drug induced neutropenia, immuneneutropenias, idiopathic neutropenia, and following infections withviruses that include, but are not limited to, HIV, CMV, and parvovirus.

In other embodiments, the 15-PGDH inhibitor can be administered to asubject to enhance recovery of platelets following bone marrowtransplantation, following umbilical cord blood transplantation,following transplantation with hematopoietic stem cells, followingconventional chemotherapy, following radiation treatment, and inindividuals with neutropenias from diseases that include but are notlimited to aplastic anemia, myelodysplasia, myelofibrosis,thrombocytopenias from other bone marrow diseases, drug inducedthrombocytopenia, immune thrombocytopenia, idiopathic thrombocytopenicpurpura, idiopathic thrombocytopenia, and following infections withviruses that include, but are not limited to, HIV, CMV, and parvovirus.

In still other embodiments, the 15-PGDH inhibitor can be administered toa subject to enhance recovery of hemoglobin following bone marrowtransplantation, following umbilical cord blood transplantation,following transplantation with hematopoietic stem cells, followingconventional chemotherapy, following radiation treatment, and inindividuals with anemias from diseases that include but are not limitedto aplastic anemia, myelodysplasia, myelofibrosis, anemia from otherbone marrow diseases, drug induced anemia, immune mediated anemias,anemia of chronic disease, idiopathic anemia, and following infectionswith viruses that include, but are not limited to, HIV, CMV, andparvovirus.

In some embodiments, the 15-PGDH inhibitor can be administered to asubject to enhance numbers of bone marrow stem cell numbers followingbone marrow transplantation, following umbilical cord bloodtransplantation, following transplantation with hematopoietic stemcells, following conventional chemotherapy, following radiationtreatment, in individuals with other bone marrow diseases, inindividuals with cytopenias following viral infections, and inindividuals with cytopenias.

In other embodiments, the 15-PGDH inhibitor can be administered to asubject to enhance response to cytokines administered to individualswith cytopenias that include but are not limited to neutropenia,thrombocytopenia, lymphocytopenia, and anemia. Cytokines whose responsesmay be enhanced by SW033291 include, but are not limited to: G-CSF,GM-CSF, EPO, IL-3, IL-6, TPO, SCF, and TPO-RA (thrombopoietin receptoragonist).

In further embodiments, the 15-PGDH inhibitor can be administered to asubject or to a tissue graft of a subject to mitigate graft rejection,to enhance graft engraftment, to enhance graft engraftment followingtreatment of the subject or the marrow of the subject with radiationtherapy, chemotherapy, or immunosuppressive therapy, to conferresistance to toxic or lethal effects of exposure to radiation, conferresistance to the toxic effect of Cytoxan, the toxic effect offludarabine, the toxic effect of chemotherapy, or the toxic effect ofimmunosuppressive therapy, to decrease infection, and/or to decreasepulmonary toxicity from radiation.

In other embodiments, the 15-PGDH inhibitor can be administered to arecipient of a tissue stem cell transplant, including but not limited toa transplant with hematopoietic stem cells, neural stem stems,mesenchymal stem cells, or stem cells for other tissues, so as toaccelerate tissue regeneration and repair following the transplant.

Additionally, in a model organism, PGE₂ signaling stimulates liverregeneration and increase survival after exposure to hepatoxic agents,such as acetaminophen. Hence, 15-PGDH inhibitors described herein may beutilized to increase liver regeneration after liver resection, in othersettings that include after liver surgery, after live liver donation, orafter receiving a liver transplant or to increase liver regeneration andincrease survival after exposures to hepatoxic agents, including but notlimited to acetaminophen and similar compounds.

PGE1 analogues have also been used in the treatment of erectiledysfunction. Accordingly, in some embodiments, 15-PGDH inhibitorsdescribed herein can used either alone or combination with aprostaglandin for the treatment of erectile dysfunction.

It will be appreciated that the other 15-PGDH inhibitors can be used inthe methods described described herein. These other 15-PGDH inhibitorscan include known 15-PGDH inhibitors including, for example, tetrazolecompounds of formulas (I) and (II), 2-alkylideneaminooxyacetamidecompounds of formula (I), heterocyclic compounds of formulas (VI) and(VII), and pyrazole compounds of formula (III) described in U.S. PatentApplication Publication No. 2006/0034786 and U.S. Pat. No. 7,705,041;benzylidene-1,3-thiazolidine compounds of formula (I) described in U.S.Patent Application Publication No. 2007/0071699;phenylfurylmethylthiazolidine-2,4-dione andphenylthienylmethylthiazolidine-2,4-dione compounds described in U.S.Patent Application Publication No. 2007/0078175; thiazolidenedionederivatives described in U.S. Patent Application Publication No.2011/0269954; phenylfuran, phenylthiophene, or phenylpyrrazole compoundsdescribed in U.S. Pat. No. 7,294,641, 5-(3,5-disubstitutedphenylazo)-2-hydroxybenzene-acetic acids and salts and lactonesdescribed in U.S. Pat. No. 4,725,676, and azo compounds described inU.S. Pat. No. 4,889,846.

The 15-PGDH inhibitors described herein can be provided in apharmaceutical composition or cosmetic composition depending on thepathological or cosmetic condition or disorder being treated. Apharmaceutical composition containing the 15-PGDH inhibitors describedherein as an active ingredient may be manufactured by mixing thederivative with a pharmaceutically acceptable carrier(s) or anexcipient(s) or diluting the 15-PGDH inhibitors with a diluent inaccordance with conventional methods. The pharmaceutical composition mayfurther contain fillers, anti-cohesives, lubricants, wetting agents,flavoring agents, emulsifying agents, preservatives and the like. Thepharmaceutical composition may be formulated into a suitable formulationin accordance with the methods known to those skilled in the art so thatit can provide an immediate, controlled or sustained release of the15-PGDH inhibitors after being administered into a mammal.

In some embodiments, the pharmaceutical composition may be formulatedinto a parenteral or oral dosage form. The solid dosage form for oraladministration may be manufactured by adding excipient, if necessary,together with binder, disintegrants, lubricants, coloring agents, and/orflavoring agents, to the 15-PGDH inhibitors and shaping the resultingmixture into the form of tablets, sugar-coated pills, granules, powderor capsules. The additives that can be added in the composition may beordinary ones in the art. For example, examples of the excipient includelactose, sucrose, sodium chloride, glucose, starch, calcium carbonate,kaolin, microcrystalline cellulose, silicate and the like. Exemplarybinders include water, ethanol, propanol, sweet syrup, sucrose solution,starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl starch, methylcellulose, ethylcellulose,shellac, calcium phosphonate and polypyrrolidone. Examples of thedisintegrant include dry starch, sodium arginate, agar powder, sodiumbicarbonate, calcium carbonate, sodium lauryl sulfate, stearicmonoglyceride and lactose. Further, purified talc, stearates, sodiumborate, and polyethylene glycol may be used as a lubricant; and sucrose,bitter orange peel, citric acid, tartaric acid, may be used as aflavoring agent. In some embodiments, the pharmaceutical composition canbe made into aerosol formulations (e.g., they can be nebulized) to beadministered via inhalation.

The 15-PGDH inhibitors described herein may be combined with flavoringagents, buffers, stabilizing agents, and the like and incorporated intooral liquid dosage forms such as solutions, syrups or elixirs inaccordance with conventional methods. One example of the buffers may besodium citrate. Examples of the stabilizing agents include tragacanth,acacia and gelatin.

In some embodiments, the 15-PGDH inhibitors described herein may beincorporated into an injection dosage form, for example, for asubcutaneous, intramuscular or intravenous route by adding thereto pHadjusters, buffers, stabilizing agents, relaxants, topical anesthetics.Examples of the pH adjusters and the buffers include sodium citrate,sodium acetate and sodium phosphate. Examples of the stabilizing agentsinclude sodium pyrosulfite, EDTA, thioglycolic acid and thiolactic acid.The topical anesthetics may be procaine HCl, lidocaine HCl and the like.The relaxants may be sodium chloride, glucose and the like.

In other embodiments, the 15-PGDH inhibitors described herein may beincorporated into suppositories in accordance with conventional methodsby adding thereto pharmaceutically acceptable carriers that are known inthe art, for example, polyethylene glycol, lanolin, cacao butter orfatty acid triglycerides, if necessary, together with surfactants suchas Tween.

The pharmaceutical composition may be formulated into various dosageforms as discussed above and then administered through various routesincluding an oral, inhalational, transdermal, subcutaneous, intravenousor intramuscular route. The dosage can be a pharmaceutically effectiveamount. The pharmaceutically effective amount can be an amount of the15-PGDH inhibitor to treat or improve alopecia, cardiovascular disease,gastrointestinal disease, wounds, and renal disease. Thepharmaceutically effective amount of the compound will be appropriatelydetermined depending on the kind and the severity of the disease to betreated, age, sex, body weight and the physical condition of thepatients to be treated, administration route, duration of therapy andthe like. Generally, the effective amount of the compound may be in therange of about 1 to 1,000 mg in the oral administration, about 0.1 to500 mg in the intravenous administration, about 5 to 1,000 mg in therectal administration. Generally, the daily dosage for adults is in therange of about 0.1 to 5,000 mg, preferably about to 1,000 mg but cannotbe determined uniformly because it depends on age, sex, body weight andthe physical condition of the patients to be treated. The formulationmay be administered once a day or several times a day with a divideddose.

Cosmetic compositions containing the 15-PGDH inhibitor can include anysubstance or preparation intended to be brought into contact with thevarious superficial parts of the human body (epidermis, body hair andhair system, nails, lips and external genital organs) or with the teethor the buccal mucous membranes for the purpose, exclusively or mainly,of cleansing them, of giving them a fragrance, of modifying theirappearance and/or of correcting body odors and/or protecting them or ofmaintaining them in good condition.

The cosmetic composition can comprise a cosmetically acceptable mediumthat may be water or a mixture of water and at least one solventselected from among hydrophilic organic solvents, lipophilic organicsolvents, amphiphilic organic solvents, and mixtures thereof.

For topical application, the cosmetic composition can be administered inthe form of aqueous, alcoholic, aqueous-alcoholic or oily solutions orsuspensions, or of a dispersion of the lotion or serum type, ofemulsions that have a liquid or semi-liquid consistency or are pasty,obtained by dispersion of a fatty phase in an aqueous phase (O/W) orvice versa (W/O) or multiple emulsions, of a free or compacted powder tobe used as it is or to be incorporated into a physiologically acceptablemedium, or else of microcapsules or microparticles, or of vesiculardispersions of ionic and/or nonionic type. It may thus be in the form ofa salve, a tincture, milks, a cream, an ointment, a powder, a patch, animpregnated pad, a solution, an emulsion or a vesicular dispersion, alotion, aqueous or anhydrous gels, a spray, a suspension, a shampoo, anaerosol or a foam. It may be anhydrous or aqueous. It may also comprisesolid preparations constituting soaps or cleansing cakes.

The cosmetic compositions may in particular comprise a hair carecomposition, and in particular a shampoo, a setting lotion, a treatinglotion, a styling cream or gel, restructuring lotions for the hair, amask, etc. The cosmetic compositions can be a cream, a hair lotion, ashampoo or a conditioner. These can be used in particular in treatmentsusing an application that may or may not be followed by rinsing, or elsein the form of a shampoo. A composition in the form of a foam, or elsein the form of spray or an aerosol, then comprising propellant underpressure, is also intended. It can thus be in the form of a lotion,serum, milk, cream, gel, salve, ointment, powder, balm, patch,impregnated pad, cake or foam.

In particular, the compositions for application to the scalp or the haircan be in the form of a hair care lotion, for example for daily ortwice-weekly application, of a shampoo or of a hair conditioner, inparticular for twice-weekly or weekly application, of a liquid or solidsoap for cleansing the scalp, for daily application, of a hairstyleshaping product (lacquer, hair setting product or styling gel), of atreatment mask, or of a foaming gel or cream for cleansing the hair.These may also be in the form of a hair dye or mascara to be appliedwith a brush or a comb.

Moreover, for topical application to the eyelashes or body hair, thecompositions may be in the form of a pigmented or unpigmented mascara,to be applied with a brush to the eyelashes or alternatively to beard ormoustache hair. For a composition administration by injection, thecomposition may be in the form of an aqueous lotion or an oilysuspension. For oral use, the composition may be in the form ofcapsules, granules, oral syrups or tablets. According to a particularembodiment, the composition is in the form of a hair cream or hairlotion, a shampoo, a hair conditioner or a mascara for the hair or forthe eyelashes.

In a known manner, the cosmetic compositions may also contain adjuvantsthat are normal in the cosmetics field, such as hydrophilic orlipophilic gelling agents, hydrophilic or lipophilic additives,preservatives, antioxidants, solvents, fragrances, fillers, UV-screeningagents, odor absorbers and dyestuffs. The amounts of these variousadjuvants are those conventionally used in the cosmetics field, and arefor example from 0.1% to 20%, in particular less than or equal to 10%,of the total weight of the composition. According to their nature, theseadjuvants can be introduced into the fatty phase, into the aqueous phaseand/or into the lipid spherules.

In some embodiments, the 15-PGDH inhibitor can be administered in acombinatorial therapy or combination therapy that includesadministration of a 15-PGDH inhibitor with one or more additional activeagents. The phrase “combinatorial therapy” or “combination therapy”embraces the administration of the 15-PGDH inhibitor, and one or moretherapeutic agents as part of a specific treatment regimen intended toprovide beneficial effect from the co-action of these therapeuticagents. Administration of these therapeutic agents in combinationtypically is carried out over a defined period (usually minutes, hours,days or weeks depending upon the combination selected). “Combinatorialtherapy” or “combination therapy” is intended to embrace administrationof these therapeutic agents in a sequential manner, that is, whereineach therapeutic agent is administered at a different time, as well asadministration of these therapeutic agents, or at least two of thetherapeutic agents, in a substantially simultaneous manner.Substantially simultaneous administration can be accomplished, forexample by administering to the subject an individual dose having afixed ratio of each therapeutic agent or in multiple, individual dosesfor each of the therapeutic agents. Sequential or substantiallysimultaneous administration of each therapeutic agent can be effected byany appropriate route including, but not limited to, oral routes,intravenous routes, intramuscular routes, and direct absorption throughmucous membrane tissue. The therapeutic agents can be administered bythe same route or by different routes. The sequence in which thetherapeutic agents are administered is not narrowly critical.

In some embodiments, the additional active agent can be chosen inparticular from lipoxygenase inhibitors as described in EP 648488, thebradykinin inhibitors described in particular in EP 845700,prostaglandins and their derivatives, in particular those described inWO 98/33497, WO 95/11003, JP 97-100091, JP 96-134242, the agonists orantagonists of the receptors for prostaglandins, and the nonprostanoicanalogues of prostaglandins as described in EP 1175891 and EP 1175890,WO 01/74307, WO 01/74313, WO 01/74314, WO 01/74315 or WO 01/72268.

In other embodiments, the 15-PGDH inhibitors can be administered incombination with active agents, such as vasodilators, prostanoidagonists, antiandrogens, cyclosporins and their analogues,antimicrobials, triterpenes, alone or as a mixture. The vasodilators caninclude potassium channel agonists including minoxidil and itsderivatives, aminexil and the compounds described in U.S. Pat. Nos.3,382,247, 5,756,092, 5,772,990, 5,760,043, 5,466,694, 5,438,058,4,973,474, chromakalin and diazoxide. The antiandrogens can include5.alpha.-reductase inhibitors such as finasteride and the compoundsdescribed in U.S. Pat. No. 5,516,779, cyproterone acetate, azelaic acid,its salts and its derivatives, and the compounds described in U.S. Pat.No. 5,480,913, flutamide and the compounds described in U.S. Pat. Nos.5,411,981, 5,565,467 and 4,910,226. The antimicrobial compounds caninclude selenium derivatives, ketoconazole, triclocarban, triclosan,zinc pyrithione, itraconazole, pyridine acid, hinokitiol, mipirocine,and the compounds described in EP 680745, clinycine hydrochloride,benzoyl or benzyl peroxide and minocycline. The anti-inflammatory agentscan include inhibitors specific for Cox-2 such as for example NS-398 andDuP-697 (B. Batistini et al., DN&P 1994; 7(8):501-511) and/or inhibitorsof lipoxygenases, in particular 5-lipoxygenase, such as for examplezileuton (F. J. Alvarez & R. T. Slade, Pharmaceutical Res. 1992;9(11):1465-1473).

Other active compounds, which can be present in pharmaceutical and/orcosmetic compositions can include aminexil and its derivatives,60-[(9Z,12Z)octadec-9,12-dienoyl]hexapyranose, benzalkonium chloride,benzethonium chloride, phenol, oestradiol, chlorpheniramine maleate,chlorophyllin derivatives, cholesterol, cysteine, methionine, benzylnicotinate, menthol, peppermint oil, calcium panthotenate, panthenol,resorcinol, protein kinase C inhibitors, prostaglandin H synthase 1 orCOX-1 activators, or COX-2 activators, glycosidase inhibitors,glycosaminoglycanase inhibitors, pyroglutamic acid esters,hexosaccharidic or acylhexosaccharidic acids, substituted ethylenearyls,N-acylated amino acids, flavonoids, derivatives and analogues ofascomycin, histamine antagonists, triterpenes, such as ursolic acid andthe compounds described in U.S. Pat. Nos. 5,529,769, 5,468,888,5,631,282, saponins, proteoglycanase inhibitors, agonists andantagonists of oestrogens, pseudopterins, cytokines and growth factorpromoters, IL-1 or IL-6 inhibitors, IL-10 promoters, TNF inhibitors,vitamins, such as vitamin D, analogues of vitamin B12 and panthotenol,hydroxy acids, benzophenones, esterified fatty acids, and hydantoin.

Pharmaceutical and/or cosmetic compositions including the 15-PGDHinhibitor described herein can additionally contain, for example, atleast one compound chosen from prostaglandins, in particularprostaglandin PGE₁, PGE₂, their salts, their esters, their analogues andtheir derivatives, in particular those described in WO 98/33497, WO95/11003, JP 97-100091, JP 96-134242, in particular agonists of theprostaglandin receptors. It may in particular contain at least onecompound such as the agonists (in acid form or in the form of aprecursor, in particular in ester form) of the prostaglandin F₂αreceptor, such as for example latanoprost, fluprostenol, cloprostenol,bimatoprost, unoprostone, the agonists (and their precursors, inparticular the esters such as travoprost) of the prostaglandin E₂receptors such as 17-phenyl PGE₂, viprostol, butaprost, misoprostol,sulprostone, 16,16-dimethyl PGE₂, 11-deoxy PGE₁, 1-deoxy PGE₁, theagonists and their precursors, in particular esters, of theprostacycline (IP) receptor such as cicaprost, iloprost,isocarbacycline, beraprost, eprostenol, treprostinil, the agonists andtheir precursors, in particular the esters, of the prostaglandin D₂receptor such as BW245C((4S)-(3-[(3R,S)-3-cyclohexyl-3-isopropyl]-2,5-dioxo)-4-imidazolidinehept-anoicacid), BW246C((4R)-(3-[(3R,S)-3-cyclohexyl-3-isopropyl]-2,5-dioxo)-4-imidazolidinehept-anoicacid), the agonists and their precursors, in particular the esters, ofthe receptor for the thromboxanes A2 (TP) such as I-BOP ([1S-[1a,2a(Z),3b(1E,3S),4a]]-7-[3-[3-hydroxy-4-[4-(iodophenoxy)-1-butenyl]-7-oxabicyclo-[2.2.1]hept-2-yl]-5-heptenoicacid).

Advantageously, the composition can include at least one 15-PGDHinhibitor as defined above and at least one prostaglandin or oneprostaglandin derivative such as for example the prostaglandins ofseries 2 including in particular PGF_(2α) and PGE₂ in saline form or inthe form of precursors, in particular of the esters (example isopropylesters), their derivatives such as 16,16-dimethyl PGE₂, 17-phenyl PGE₂and 16,16-dimethyl PGF_(2α) 17-phenyl PGF_(2α), prostaglandins of series1 such as 11-deoxyprostaglandin E1, 1-deoxyprostaglandin E1 in saline orester form, is their analogues, in particular latanoprost, travoprost,fluprostenol, unoprostone, bimatoprost, cloprostenol, viprostol,butaprost, misoprostol, their salts or their esters.

The invention is further illustrated by the following examples, which isnot intended to limit the scope of the claims.

Example 1

This Example describes the activities of four compounds with respect tothe enzyme 15-Prostaglandin Dehydrogenase (15-PGDH) (encoded by the geneHPGD). The compounds are SW033291, SW054384, SW124531, SW145753 and havethe following formulas:

FIG. 1 shows that SW033291, SW054384, and SW145753 all increaseluciferase activity of cells the express a 15-PGDH luciferase fusionconstruct created by targeted gene knock-in of renilla luciferase intothe last coding exon of 15-PGDH. The activity is demonstrated in threedifferent colon cancer cell lines all engineered to contain the15-PGDH-luciferase fusion. These cell lines are Vaco-9m (V9m), LS174T,Vaco503 (V503). SW054384 is in general the best inducer, and showsmaximum activity at 6.25 μM. Value of 1.0 on the Y-axis is the basallevel of reporter activity in cells treated with drug free DMSO vehicle.

FIG. 2 shows western blots demonstrating that SW033291, SW054384, andSW145753 all increase levels of 15-PGDH protein in cell lines V503,LS174T, and V503 treated with 7.5 μM compound for 48 hours. UntreatedFET cells provide a positive control for 15-PGDH expression.

FIG. 3 shows western blot demonstrating SW124531 also increases 15-PGDHprotein levels in colon cell lines (FET cells treated with TGF-β (10ng/ml) for 48 hours are used as a positive control for 15-PGDHexpression in certain panels).

FIG. 4 shows western blot demonstrating 5 μM SW124531 increases levelsof 15-PGDH protein (wt-PGDH) expressed from a cDNA expression vector inV400-S3-2-32 cells, and also increases protein levels of a catalyticallydead mutant 15-PGDH (mu-PGDH) also expressed from a cDNA expressionvector in V400-M3-2-72 cells. As these proteins are expressed from aheterologous CMV promoter, the findings suggest that the compounds workdirectly on stabilizing the 15-PGDH protein. The compounds show noeffects on levels of a related enzyme, 17-beta-estradiol-dehydrogenase.

FIG. 5 shows increase in 15-PGDH protein levels in V503 cells treatedwith SW124531 as assayed by immuno-fluorescence (upper two rows) and bywestern blot (lower panel).

FIGS. 6-9 show that SW033291, SW054384, SW145753, and SW124531 do not ingeneral alter 15-PGDH mRNA levels in treated colon cancer cell lines asassessed by real-time PCR. The only exception is the slight increase in15-PGDH mRNA in SW033291 treated V503 cells, which is less than theinduction of 15-PGDH protein as well as 15-PGDH-luciferase reporterlevels seen in SW033291 treated V503 cells. In these studies parentalcell lines (not containing the 15-PGDH-luciferase reporter) areemployed.

FIG. 10 shows the effects of three compounds on total 15-PGDH activityin cell lines treated with the compounds. Cell lines were treated withcompounds at 7.5 μM for 48 hours, and then pelleted. Pellets were lysedand total 15-PGDH activity measured and normalized to 1,000,000 inputcells per pellet. 15-PGDH activity was assayed by measuring the transferof tritium from 15(S)-[15-3H] PGE₂ to glutamate by coupling 15-PGDH withglutamate dehydrogenase as described in (Chi X, Freeman B M, Tong M,Zhao Y, Tai H H. 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) isup-regulated by flurbiprofen and other non-steroidal anti-inflammatorydrugs in human colon cancer HT29 cells. Arch Biochem Biophys. 2009;487(2):139-45). Activity is measured as pmol PGE₂/min/million cells. Asshown, SW033291 markedly inhibits 15-PGDH activity in all three of thecell lines tested. We conclude that although SW033291 increases total15-PGDH protein levels in cells, it also inactivates 15-PGDH enzymeactivity.

In contrast, 15-PGDH enzyme activity is increased in cells treated withSW054384 and in cells treated with SW145753.

FIG. 11 shows the effect on activity of recombinant 15-PGDH protein (a15-PGDH-GST fusion protein) incubated with varying concentrations of thetest compounds, with 15-PGDH activity across a range of compoundconcentrations recorded on the table and displayed on the correspondinggraphs. As shown, SW033291 is a potent inhibitor of 15-PGDH activity,with an IC₅₀ of <1.25 nM. This contrasts with the IC₅₀ of between 25nM-62.5 nM measured for the commercial 15-PGDH inhibitor available fromCayman Chemical (Cayman catalogue item 10638, Cayman Chemical number13695).

FIG. 12 shows repeat testing of the effects of SW033291 and SW054384 onactivity of recombinant 15-PGDH protein tested in vitro. Assays weredone by measuring the transfer of tritium from 15(S)-[15-3H] PGE₂ toglutamate (at 1 μM PGE₂ substrate) shown at left (panels A, C), or bydirect fluorescence monitoring of NADH generation by 15-PGDH (done at 20μM PGE₂ substrate) shown at right (panels B, D). SW033291 is againconfirmed as a highly potent 15-PGDH inhibitor with an IC50 of 0.7 nM asmeasured in the tritium assay and an IC₅₀ of 1.6 nM as measured in thefluorescence assay. The relative insensitivity of the IC50 to substrateconcentration suggests that SW033291 is a non-competitive inhibitor of15-PGDH.

FIG. 13 shows results of assays of 15-PGDH activity using the tritiummethod in cells treated with SW124531 (upper panel) and in recombinant15-PGDH protein treated with SW124531 (lower panel). SW124531 showsactivity in increasing 15-PGDH activity in most cell lines, though thisactivity is best in cell lines in which basal 15-PGDH activity is >10units. SW124531 also inhibits activity of 15-PGDH recombinant protein atan IC₅₀ of 50 nM.

FIG. 14 shows assay of different compounds for ability to directly bindto recombinant 15-PGDH protein as measured by shifting the meltingtemperature of the protein. The melting of the protein is followed bymeasurement of the fluorescence of SYPRO Orange dye (Sigma #55692) thatincreases as the dye binds to hydrophobic residues exposed as theprotein melt. The graph at upper left shows the melt curves of 15-PGDHwith all of the assays done in the presence of the different compoundssuperimposed on each other. The graph at upper right plots the negativederivative of fluorescence versus temperature for each of the curvesshown at left, with the melting point measured as the temperature of thenegative peak (i.e., the point of most rapid change in the fluorescenceversus temperature plot). The results are shown in tabular form on thetable below. Lapatinib is used as a negative control. There is nobinding of any drug in the absence of enzyme co-factor (either NAD orNADH). In the presence of either NAD or NADH, SW033291 creates two peaksin the melting curve, with one of these peaks displaced by 15 degreesCelsius, consistent with SW033291 binding directly to 15-PGDH. SW124531and SW145753 also show evidence of direct binding to 15-PGDH. In thisassay, SW054384 cannot be demonstrated to bind 15-PGDH. It is possiblethat SW054384 does bind to 15-PGDH, but that the binding is weak and ismelted off at a temperature below the melting temperature of the 15-PGDHprotein. Assays were done at both 10 μM and 100 μM cofactor (testingboth NAD and NADH), which compares well with the published Km of NAD of15.8 μM.

FIG. 15 shows that none of the four compounds tested induce a shift inthe melting temperature of catalytically inactive mutant 15-PGDHprotein. We interpret the induction of 15-PGDH mutant protein bySW124531 as suggesting that SW124531 likely has weak binding to mutant15-PGDH that is able to stabilize protein at 37° C., but with the drugmelted off at a temperature below 50° C., that is the meltingtemperature of the protein.

FIG. 16 shows the in vivo modulation by compounds of 15-PGDH activity asreflected in PGE₂ levels that are assayed in the medium of A549 cellsthat have been stimulated by IL1-beta for 23 hours, with compound addedfor the last 5 hours (blue bars). The increment in PGE₂ level shows theclear inhibition of 15-PGDH activity in the cells by addition ofSW033291 (as well as SW145753, SW124531, and a commercial 15-PGDHinhibitor from Cayman Chemical. In an additional iteration (redbars)(2), SW054384 was added commencing 24 hours before addition ofIL1-beta, and then maintained for the next 26 hours in the presence ofIL1-beta. The lower level of PGE₂ produced supports that in these cellsSW054384 increased the 15-PGDH activity. Panel at left shows raw data;whereas, panel at right shows data normalized for cell numbers presentat end of the experiment. PGE₂ levels are assayed by ELISA.

FIG. 17 shows the dose response of effect on SW033291 on PGE₂ productionfrom IL1-beta treated A549 cells, as reflected in PGE₂ levels that areassayed in the medium of A549 cells that have been stimulated byIL1-beta for 24 hours, with SW033291 added for the last 8 hours.

FIG. 18 shows the in vivo modulations by 2.5 μM compounds of 15-PGDHactivity as reflected in PGE₂ levels following addition of PGE₂ into themedium of Vaco-503 cells. In this study cells are treated with compoundfor 24 hours after which PGE₂ is added into the medium. After an added24 hours PGE₂ levels remaining in the medium are assayed by Elisa. Datalabeled “medium” is a control lane with PGE₂ added to medium alone, inthe absence of cells. Data labeled DMSO is a control in which cells aretreated with DMSO only (the vehicle for the compounds). The differencebetween the “medium” and the “DMSO” lanes represents the cell dependentdegradation of PGE₂ by 15-PGDH. Again demonstrated, is the near completeblockade of 15-PGDH activity by addition of 2.5 μM SW033291, asreflected by the blockade in PGE₂ degradation. Additionally demonstratedis the stimulation of 15-PGDH activity by SW054384, as reflected by theincreased degradation of PGE₂.

FIG. 19 shows the activity of 2.5 μM SW033291 in speeding the healing ofa model wound consisting of a scratch in a monolayer of HaCaT cellsobserved over 48 hours of treatment. TGF-beta serves as the positivecontrol in the assay.

FIG. 20 shows the quantitation of the width of the scratch at 0 and 48hours in the control, 2.5 μM SW033291 treated cells, and the TGF-beta (1ng/ml) treated cells.

Example 2 Analysis of Analogues of Lead Compounds SW033291, a 15-PGDHInhibitor

This Example provides data on a group of structural analogues ofSW033291. Data provided is the IC50 of each compound for inhibitingenzymatic activity of recombinant 15-PGDH in an in vitro assay.Recombinant 15-PGDH is human unless otherwise specified.

TABLE 1 Enzyme Inhibitor IC₅₀ (nM) at 5 nM Structure/Smiles ID # 15-PGDH

SW033290 525.40 CS(═O)(═O)C1═C(N)C2═C(S1)N═C(C═C2C1═CC═CC═C1)C1═CC═CS1

SW033291 2.53 CCCCS(═O)C1═C(N)C2═C(S1)N═C(C═C2C1═CC═CC═C1)C1═CC═CS1

SW033291 (Rat PGDH) 2.74CCCCS(═O)C1═C(N)C2═C(S1)N═C(C═C2C1═CC═CC═C1)C1═CC═CS1

SW033291 (Mouse PGDH) 2.56CCCCS(═O)C1═C(N)C2═C(S1)N═C(C═C2C1═CC═CC═C1)C1═CC═CS1

SW206976 >7500 CCCCC(═O)C1═C(N)C2═C(S1)N═C(C═C2C1═CC═CC═C1)C1═CC═CS1

SW206977 >7500 CCCNC(═O)C1═C(N)C2═C(S1)N═C(C═C2C1═CC═CC═C1)C1═CC═CS1

SW206978 >7500 CCOC(═O)C1═C(N)C2═C(S1)N═C(C═C2C1═CC═CC═C1)C1═CC═CS1

SW206979 >7500 NC1═C(SC2═C1C(═CC(═N2)C1═CC═CS1)C1═CC═CC═C1)C(O)═O

SW206980 0.97 CCCCS(═O)C1═C(N)C2═C(S1)N═C(C═C2)C1═CC═CS1

SW206992 1.41 CCCCS(═O)C1═C(N)C2═CC═C(N═C2S1)C1═NC═CS1

SW208064 151.40 CCCCS(═O)C1═C(N)C2═C(S1)N═CC═C2

SW208065 4.87 CCCCS(═O)C1═C(N)C2═C(S1)N═C(N═C2C1═CC═CC═C1)C1═CC═CC═C1

SW208066 1.37 CCCCS(═O)C1═C(N)C2═C(S1)N═C(C═C2C1═CC═CC═C1)C1═NC═CS1

SW208067 2.40 CCCCS(═O)C1═C(N)C2═C(S1)N═C(N═C2C1═CC═CC═C1)C1═CC═CS1

SW208068 >7500 CCCCSC1═NC═CC═C1N

SW208069 >7500 CCCCS(═O)C1═NC═CC═C1[N+]([O−])═O

SW208070 >7500 CCCCS(═O)C1═NC═CC═C1N

SW208199 2.9 CCCCS(═O)C1═C(N)C2═C(S1)N═C(C═C2C1═CC═C(Br)C═C1)C1═CC═CS1

SW208078 25.40 CCCCS(═O)(═O)C1═C(N)C2═C(S1)N═C(C═C2C1═CC═CC═C1)C1═CC═CS1

SW208080 2.61 CCCCS(═O)C1═C(N)C2═C(C═C(N═C2S1)C1═CC═CS1)C1═CC═CC═C1

SW208081 17.85 CCCCCCS(═O)C1═C(N)C2═C(C═C(N═C2S1)C1═CC═CS1)C1═CC═CC═C1

SW208079 124.90 C[S+]([O−])C1═C(N)C2═C(C═C(N═C2S1)C1═CC═CS1)C1═CC═CC═C1

SW211667 (33291 S isomer) 195.30CCCC[S@+]([O−])C1═C(N)C2═C(C═C(N═C2S1)C1═CC═CS1)C1═CC═CC═C1

SW211668 (33291 R isomer) 1.34CCCC[S@+]([O−])C1═C(N)C2═C(C═C(N═C2S1)C1═CC═CS1)C1═CC═CC═C1

SW208430 >2500 CCCCSC1═CC2═CC═C(N═C2S1)C1═CC═CC═C1

SW208432 3.53 CCCCS(═O)C1═CC2═CC═C(N═C2S1)C1═CC═CC═C1

SW208434 >2500 CCCCS(═O)(═O)C1═CC2═CC═C(N═C2S1)C1═CC═CC═C1

SW208435 >2500 CCCCSC1═C(N)C2═CC═C(N═C2S1)C1═CC═CC═C1

SW208436 1.14 CCCCS(═O)C1═C(N)C2═C(N═C(N═C2S1)C1═NC═CS1)C1═CC═CC═C1

SW208437 2.05 CCCS(═O)C1═C(N)C2═C(C═C(N═C2S1)C1═CC═CS1)C1═CC═CC═C1

SW208438 3.16 CC(C)S(═O)C1═C(N)C2═C(C═C(N═C2S1)C1═CC═CS1)C1═CC═CC═C1

SW208488 1.43 CCCCS(═O)C1═C(N)C2═C(C)C═C(N═C2S1)C1═CC═CS1

SW208494 >7500 CCCCSC1═NC2═NC(═CC═C2N1)C1═CC═CS1

SW208495 >7500 CCCCS(═O)C1═NC2═NC(═CC═C2N1)C1═CC═CS1

SW208496 1.37 CCCCS(═O)C1═C(N)C2═C(S1)N═C(C═C2C1═CC═CC═C1)C1═NC═CO1

SW208599 1230.00 CCCCS(═O)C1═NC2═CC═C(N═C2S1)C1═CC═CS1

SW208660 2.86 CC(C)S(═O)C1═C(N)C2═C(S1)N═C(C═C2C1═CC═CC═C1)C1═NC═CO1

SW208661 5.27 CCCS(═O)C1═C(N)C2═C(S1)N═C(C═C2C)C1═NC═CS1

SW208662 1319.00 CCCCS(═O)C1═CC2═C(S1)N═C(N═C2N1CCCCC1)C1═CC═CC═C1

SW208663 135.50 CCCS(═O)C1═CN)C2═C(S1)N═C(C═C2C)N1CCOCC1

SW208664 7.62 CC(C)S(═O)C1═C(N)C2═C(S1)N═C(C═C2C)C1═NC-CS1

SW208776 93.29 CCCCS(═O)C1═CC2═C(N═C(N═C2S1)C1═CC═CC═C1)C1═CC═CC═C1

SW208777 2.65 CCCCS(═O)C1═C(N)C2═C(C═C(N═C2S1)C1═NC═CS1)C1═CC═CN═C1

SW208778 3.99 CC(C)S(═O)C1═C(N)C2═C(C═C(N═C2S1)C1═NC═CS1)C1═CC═CN═C1

SW208780 5.55 CC(C)S(═O)C1═C(N)C2═C(C═C(N═C2S1)C1═NC═CS1)C1═CC═CC═C1

SW208781 1.99 CCCS(═O)C1═C(N)C2═C(C═C(N═C2S1)C1═CC═CS1)C(═O)OCC

SW208782 70.91 CCCS(═O)C1═C(N)C2═C(C═C(N═C2S1)C1═CC═CS1)C(O)═O

SW209123 40.80CCCCS(═O)C1═C(N)C2═C(C3═CC═C(Br)C═C3)C(C(═O)OCC)═C(N═C2S1)C1═NC═CS1

SW209124 1.83 CCCCS(═O)C1═C(N)C2═C(C═C(N═C2S1)C1═NC═CS1)C1═NC═CS1

SW209125 2.13 CCCCS(═O)C1═C(N)C2═C(C═C(N═C2S1)C1═NC═CS1)C1═NC═CN1C

SW209126 6.67 CCCCS(═O)C1═C(N)C2═C(C═C(N═C2S1)C1═NC═CN1C)C1═CC═CC═C1

SW209127 15.13CCCCS(═O)C1═C(N)C2═C(C═C(N═C2S1)C1═NC═CS1)C1═CC═C(C═C1)C(═O)OC

SW209128 4914.00CC(═O)OCCCCS(═O)(═O)C1═C(N)C2═C(C═C(N═C2S1)C1═CC═CS1)C1═CC═CC═C1

SW209129 41.52CC(═O)OCCCC[S+]([O−])C1═C(N)C2═C(C═C(N═C2S1)C1═CC═CS1)C1═CC═CC═C1

SW209271 34.18NC1═C(SC2═NC(═CC(═C12)C1═CC═CC═C1)C1═CC═CS1)[S+]([O−])CCCCO

SW209272 530.20CC(═O)OCCCS(═O)(═O)C1═C(N)C2═C(C═C(N═C2S1)C1═CC═CS1)C1═CC═CC═C1

SW209273 21.62CC(═O)OCCC[S+]([O−])C1═C(N)C2═C(C═C(N═C2S1)C1═CC═CS1)C1═CC═CC═C1

SW209274 36.57NC1═C(SC2═NC(═CC(═C12)C1═CC═CC═C1)C1═CC═CS1)[S+]([O−])CCCO

SW209275 80.02 CCCCSC1═C(N)C2═C(C═C(N═C2S1)C1═CC═CS1)C1═CC═CC═C1

SW209276 2.09COCCC[S+]([O−])C1═C(N)C2═C(C═C(N═C2S1)C1═CC═CS1)C1═CC═CC═C1

SW209277 3.71 CCCCS(═O)C1═C(N)C2═C(S1)N═C(N═C2C1═CC═CC═C1)C1═NC═CN1C

SW209278 2.56 CCCCS(═O)C1═C(N)C2═C(S1)N═C(N═C2C1═CC═CC═C1)C1═COC═N1

SW209279 4.18 CC(C)S(═O)C1═C(N)C2═C(S1)N═C(C═C2C1═NC═CN1C)C1═NC═CS1

SW209280 3.73 CCCS(═O)C1═C(N)C2═C(S1)N═C(C═C2C1═NC═CN1C)C1═NC═CS1

SW209281 3.40CCCCS(═O)C1═C(N)C2═C(S1)N═C(C═C2C1═CC═C(C═C1)C(O)═O)C1═NC═CS1

SW209282 5.87CCCCS(═O)C1═C(N)C2═C(S1)N═C(C═C2C1═CC═C(C═C1)C(═O)N(C)C)C1═NC═CS1

SW209283 14.70 CCCCS(═O)C1═C(N)C2═C(S1)N═C(C═C2C(═O)N(C)C)C1═CC═CS1

SW209329 4.36NC1═C(SC2═NC(═CC(═C12)C1═CC═CC═C1)C1═CC═CS1)[S+]([O−])CCCCC1

SW209330 4.01NC1═C(SC2═NC(═CC(═C12)C1═CC═CC═C1)C1═CC═CS1)[S+]([O−])CCCC1

SW209331 6.14 NC1═C(SC2═NC(═CC(═C12)C1═CC═CC═C1)C1═CC═CS1)[S+]([O−])CCCF

SW209332 8.72NC1═C(SC2═NC(═CC(═C12)C1═CC═CC═C1)C1═CC═CS1)[S+]([O−])CCCC#N

SW209333 >2500CCCCS(═O)(═O)C1═C(N)C2═C(C═C(N═C2S1)C1═NC═CS1)C1═CC═C(CO)C═C1

SW209415 2.60 CCCCS(═O)C1═C(N)C2═C(C═C(N═C2S1)C1═NC═CS1)C1═CN═C(C)N1C

SW209416 8.98CCCCS(═O)C1═C(N)C2═C(C═C(N═C2S1)C1═NC═CS1)C1═CC═CC(═C1)C(═O)OC

SW209417 18.50CCCCS(═O)C1═C(N)C2═C(C═C(N═C2S1)C1═NC═CS1)C1═CC═CC(═C1)C(═O)N(C)C

SW209418 4.29 CCCCS(═O)C1═C(N)C2═C(C═C(N═C2S1)C1═NC═CS1)C1═CC═CC(CO)═C1

SW209419 20.20CCCCS(═O)C1═C(N)C2═C(C═C(N═C2S1)C1═NC═CS1)C1═CC═CC(═C1)C(O)═O

SW209420 36.40CCCCS(═O)C1═C(N)C2═C(C═C(N═C2S1)C1═NC═CS1)C1═CC═CC(═C1)C(═O)N1CCN(C)CC1

SW209427 1485.00 CCCCS(═O)CSC1═NC(═CC(C2═NC═CN2C)═C1C#N)C1═NC═CS1

SW209428 2.30 CCCCS(═O)C1═C(N)C2═C(C═C(N═C2S1)C1═NC═CS1)C1═CN═C(C)N1

SW209508 2.50CCCCS(═O)C1═C(N)C2═C(S1)N═C(C═C2C1═CC═CC(═C1)C(═O)NCC═C)C1═NC═CS1

SW209509 19.30CCCCS(═O)C1═C(N)C2═C(S1)N═C(C═C2C1═CC═CC(═C1)C(═O)NCCN(C)C)C1═NC═CS1

SW209510 2.50 CCCCS(═O)C1═C(N)C2═C(S1)N═C(C═C2C1═CC═C(CO)C═C1)C1═NC═CS1

SW209511 2.50CCCCS(═O)C1═C(N)C2═C(S1)N═C(C═C2C1═CC═C(COC(C)═O)C═C1)C1═NC═CS1

SW209513 5.70CCCCS(═O)C1═C(N)C2═C(S1)N═C(C═C2C1═CC═C(CN(C)C)C═C1)C1═NC═CS1

SW211535 3.45CCCCS(═O)C1═C(N)C2═C(C═C(N═C2S1)C1═NC═CS1)C1═CC═CC(═C1)C(═O)NCCO

SW212344 20.53CCCCS(═O)C1═C(N)C2═C(C═C(N═C2S1)C1═NC═CS1)C1═CN═C(C(C)C)N1C

SW212345 2.99CCCCS(═O)C1═C(N)C2═C(C═C(N═C2S1)C1═NC═CS1)C1═CN═C(C2CC2)N1C

SW212363 2.71CCCCS(═O)C1═C(N)C2═C(S1)N═C(C═C2C1═CC═C(OCC(O)═O)C═C1)C1═NC═CS1

SW212364 3.97CCCCS(═O)C1═C(N)C2═C(C═C(N═C2S1)C1═NC═CS1)C1═CC═C(OCCO)C═C1

SW212365 8.53CCCCS(═O)C1═C(N)C2═C(C═C(N═C2S1)C1═NC═CS1)C1═CC═C(OCC(═O)OC)C═C1

SW212366 3.48CCCCS(═O)C1═C(N)C2═C(C═C(N═C2S1)C1═NC═CS1)C1═CC═C(COC(═O)CN(C)C)C═C1

SW211688 3.33 COCCCS(═O)C1═C(N)C2═C(S1)N═C(C═C2C1═CN═C(C)N1C)C1═NC═CS1

SW211689 4.06 COCCS(═O)C1═C(N)C2═C(S1)N═C(C═C2C1═CN═C(C)N1C)C1═NC═CS1

SW209415 (−)-isomer 165.00COCCS(═O)C1═C(N)C2═C(C═C(N═C2S1)C1═NC═CS1)C1═CN═C(C)N1C

SW209415 (+)-isomer 1.30CCCCS(═O)C1═C(N)C2═C(C═C(N═C2S1)C1═NC═CS1)C1═CN═C(C)N1C

We first note that the 15-PGDH inhibitory activities of SW033291 andSW209415 are at least 98% due to the activity of the (+) optical isomersof these compounds. For SW033291, the (+)-isomer is the (R) enantiomerwhereas the absolute configuration of (+)-SW209415 has not beenestablished.

Example 3

The following Example describes the synthesis of SW033291 and analoguesthereof as well as provides mass spectrometry and NMR confirmation ofthe structures.

-   -   Concistency

3-phenyl-1-(thiophen-2-yl)prop-2-en-1-one was prepared from benzaldehydeand 1-(thiophen-2-yl)ethanone via aldol condensation using proceduredescribed by Azam (Parveen, H.; Iqbal, P. F.; Azam, A. Synth. Commu.,2008, 38, 3973). ¹H NMR (400 MHz, CDCl₃) δ 7.88-7.80 (m, 2H), 7.67 (dd,J=4.9, 1.1 Hz, 1H), 7.66-7.59 (m, 2H), 7.47-7.34 (m, 4H), 7.18 (dd,J=5.0, 3.8 Hz, 1H). ESI-MS (m/z): 215 [M+H]⁺.

4-phenyl-6-(thiophen-2-yl)-2-thioxo-1,2-dihydropyridine-3-carbonitrile.To a solution of 3-phenyl-1-(thiophen-2-yl)prop-2-en-1-one (2.34 mmol,500 mg) and cyanothioacetamide (7.0 mmol, 717 mg, 3.0 equiv.) in ethanol(7 mL), a few drops of piperidine were added. The reaction was refluxedfor 3 h. The solid that formed was collected and recrystallized fromacetic acid to give designed product in 46% isolated yield. ¹H NMR (400MHz, DMSO-d₆) δ 8.17 (d, J=3.8 Hz, 1H), 7.96 (d, J=5.0 Hz, 1H),7.74-7.62 (m, 2H), 7.54 (dd, J=5.1, 2.0 Hz, 3H), 7.31-7.19 (m, 1H), 7.01(s, 1H). ESI-MS (m/z): 295 [M+H]⁺.

2-(((butylthio)methyl)sulfinyl)-4-phenyl-6-(thiophen-2-yl)nicotinonitrile.Acetic Acid (900 μL) and hydrogen peroxide (0.57 mmol, 1.5 equiv., 30%solution in water) were added to the solution of2-(((butylthio)methyl)sulfinyl)-4-phenyl-6-(thiophen-2-yl)nicotinonitrile(0.38 mmol, 150 mg) in chloroform (900 μL). The reaction mixture wasstirring at 32° C. for 45 min. The reaction was then diluted with EtOAcand washed with saturated NaHCO₃ solution, dried over magnesium sulfate,filtered and concentrated under reduced pressure to give 153 mg ofdesigned product (98%). ¹H NMR (400 MHz, CDCl₃) δ 7.75 (dd, J=3.8, 1.1Hz, 1H), 7.66-7.57 (m, 2H), 7.58-7.51 (m, 4H), 7.47 (s, 1H), 7.16 (dd,J=5.0, 3.8 Hz, 1H), 4.74 (d, J=13.0 Hz, 1H), 4.41 (d, J=13.0 Hz, 1H),2.97 (dt, J=13.0, 8.2 Hz, 1H), 2.81 (dt, J=12.9, 7.3 Hz, 1H), 1.94-1.76(m, 2H), 1.53-1.38 (m, 2H), 0.94 (t, J=7.4 Hz, 3H). ESI-MS (m/z): 413[M+H]⁺.

SW0332912-(butylsulfinyl)-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-3-aminewas prepared using procedure describe by Kalugin (Kalugin V. E. Russian.Chem. Bull., Int. Ed., 2006, 55, 529). To the solution of4-(((butylthio)methyl)sulfinyl)-2,6-diphenylpyrimidine-5-carbonitrile(0.53 mmol, 220 mg) in DMF (0.25 M)/EtOH (0.5 M) was added KOH (0.32mmol, 18 mg, 0.6 equiv., 0.1 M in water). The reaction mixture wasstirred at 35° C. for 40 min. Once complete, the reaction was dilutedwith EtOAc and washed with 10% aq. solution of acidic acid, the organicphase was separated and aqueous layer was extracted twice with EtOAc,dried over magnesium sulfate, filtered and concentrated under reducedpressure to give 211 mg of SW0332912-(butylsulfinyl)-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-3-amine(96%). ¹H NMR (400 MHz, CDCl₃) δ 7.67-7.60 (m, 1H), 7.57-7.35 (m, 7H),7.10 (dd, J=5.0, 3.7 Hz, 1H), 4.54 (s, 2H), 3.26 (ddd, J=12.8, 9.1, 6.0Hz, 1H), 3.09 (ddd, J=12.8, 9.1, 6.6 Hz, 1H), 1.83-1.61 (m, 2H),1.53-1.38 (m, 2H), 0.93 (t, J=7.3 Hz, 3H). ESI-MS (m/z): 413 [M+H]⁺.

SW2084374-phenyl-2-(propylsulfinyl)-6-(thiophen-2-yl)thieno[2,3-b]pyridin-3-aminewas prepared in 56% isolated yield using synthetic procedures describedfor the preparation of analog SW033291. ¹H NMR (400 MHz, CDCl₃) δ 7.65(dd, J=3.8, 1.1 Hz, 1H), 7.61-7.49 (m, 4H), 7.49-7.41 (m, 3H), 7.12 (dd,J=5.0, 3.7 Hz, 1H), 3.28 (ddd, J=12.7, 8.4, 6.3 Hz, 1H), 3.07 (ddd,J=12.7, 8.6, 7.0 Hz, 1H), 1.91-1.65 (m, 2H), 1.08 (t, J=7.4 Hz, 3H).APCI-MS (m/z): 399 [M+H]⁺.

SW2084382-(isopropylsulfinyl)-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-3-aminewas prepared in 48% isolated yield using synthetic procedures describedfor the preparation of analog SW033291. ¹H NMR (400 MHz, CDCl3) δ 7.64(dd, J=3.7, 1.1 Hz, 1H), 7.58-7.47 (m, 5H), 7.47-7.39 (m, 2H), 7.10 (dd,J=5.0, 3.7 Hz, 1H), 4.59 (s, 2H), 3.38 (p, J=6.8 Hz, 1H), 1.43 (d, J=6.9Hz, 3H), 1.25 (d, J=6.8 Hz, 3H). ESI-MS (m/z): 399 [M+H]⁺.

SW2084882-(butylsulfinyl)-4-methyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-3-aminewas prepared using synthetic procedures described for the preparation ofanalog SW033291. ¹H NMR (400 MHz, CDCl₃) δ 7.55 (dd, J=3.7, 1.2 Hz, 1H),7.39 (dd, J=5.0, 1.1 Hz, 1H), 7.25-7.23 (m, 1H), 7.06 (dd, J=5.0, 3.7Hz, 1H), 5.02 (s, 2H), 3.25 (ddd, J=12.7, 9.1, 6.0 Hz, 1H), 3.08 (ddd,J=12.8, 9.2, 6.4 Hz, 1H), 2.74 (s, 3H), 1.82-1.58 (m, 2H), 1.56-1.38 (m,2H), 0.93 (t, J=7.3 Hz, 3H). ESI-MS (m/z): 351 [M+H]⁺.

SW2084962-(butylsulfinyl)-6-(oxazol-2-yl)-4-phenylthieno[2,3-b]pyridin-3-aminewas prepared using synthetic procedures described for the preparation ofanalog SW033291. ¹H NMR (400 MHz, CDCl₃) δ 7.99 (s, 1H), 7.84 (d, J=0.8Hz, 1H), 7.58-7.41 (m, 5H), 7.33 (d, J=0.8 Hz, 1H), 4.65 (s, 2H), 3.30(ddd, J=12.9, 8.8, 6.2 Hz, 1H), 3.10 (ddd, J=12.8, 8.9, 6.9 Hz, 1H),1.86-1.64 (m, 2H), 1.42-1.54 (m, 2H), 0.93 (t, J=7.4 Hz, 3H). ESI-MS(m/z): 398.1 [M+H]⁺.

SW2084366-(butylsulfinyl)-4-phenyl-2-(thiazol-2-yl)thieno[2,3-d]pyrimidin-5-aminewas prepared by synthetic procedures described for the preparation ofanalog SW208065. ¹H NMR (400 MHz, CDCl₃) δ 8.06 (dd, J=3.1, 1H),7.75-7.66 (m, 2H), 7.75-7.66 (m, 3H), 7.55 (dd, J=3.1, 1H), 4.87 (s,2H), 3.30 (ddd, J=12.8, 8.4, 6.3 Hz, 1H), 3.12 (ddd, J=12.8, 8.6, 6.9Hz, 1H), 1.85-1.65 (m, 2H), 1.55-1.40 (m, 2H), 0.95 (t, J=7.3 Hz, 3H).ESI-MS (m/z): 415.1 [M+H]⁺.

SW208432 2-(butylsulfinyl)-6-phenylthieno[2,3-b]pyridine. Acetic Acid(90 μL) and hydrogen peroxide (0.06 mmol, 1.5 equiv., 30% solution inwater) were added to the solution of2-(butylthio)-6-phenylthieno[2,3-b]pyridine (0.04 mmol, 12 mg) inchloroform (90 μL). The reaction mixture was stirring at 32° C. for 1 h.The reaction was then diluted with EtOAc and washed with saturatedNaHCO₃ solution, dried over magnesium sulfate, filtered and concentratedunder reduced pressure to give designed product in 76% isolated yield.¹H NMR (400 MHz, CDCl₃) δ 8.14 (d, J=8.4, 1H), 8.08 (d, J=8.2, 2H), 7.82(d, J=8.4, 1H), 7.58-7.36 (m, 4H), 3.30-2.73 (m, 2H), 1.90-1.62 (m, 2H),1.55-1.41 (m, 2H), 0.94 (t, J=7.3 Hz, 3H). ESI-MS (m/z): 316.1 [M+H]⁺.

SW208434. Acetic Acid (200 μL) and hydrogen peroxide (0.15 mmol, 30%solution in water) were added to the solution of2-(butylthio)-6-phenylthieno[2,3-b]pyridine (0.09 mmol, 27 mg) inchloroform (200 μL). The reaction mixture was stirring at 100° C. for 30min. The reaction was then diluted with EtOAc and washed with saturatedNaHCO₃ solution, dried over magnesium sulfate, filtered and concentratedunder reduced pressure to give designed product in 81% isolated yield.¹H NMR (400 MHz, CDCl₃) δ 8.23 (d, J=8.5 Hz, 1H), 8.09 (dd, J=8.2, 1.6Hz, 2H), 7.89 (d, J=2.1 Hz, 1H), 7.59-7.39 (m, 4H), 3.39-3.10 (m, 2H),1.92-1.68 (m, 2H), 1.54-1.27 (m, 2H), 0.91 (t, J=7.3 Hz, 3H). ESI-MS(m/z): 332.1 [M+H]⁺.

SW208430 2-(butylthio)-6-phenylthieno[2,3-b]pyridine. Phenylboronic acid(0.39 mmol, 2.0 equiv), 2-(butylthio)-6-chlorothieno[2,3-b]pyridine (50mg, 0.195 mmol, 1.0 equiv), Cesium Carbonate (0.39 mmol, 2.0 equiv.),PdCl₂dppf (10 mol %), Copper Chloride (0.195 mmol, 1.0 equiv.) wereheated in DMF at 100° C. for 12 h. After cooling to r.t. the reactionmixture was diluted with EtOAc and washed with water and next brine. Theorganic layer was dried over magnesium sulfate and the solvent wasremoved under reduced pressure. The crude product was purified by flashchromatography (Hexanes/EtOAc: 8/2) to afford designed product in 32%yield. ¹H NMR (400 MHz, CDCl₃) δ 8.09-8.00 (m, 2H), 7.93 (d, J=8.3 Hz,1H), 7.69 (d, J=8.3 Hz, 1H), 7.52-7.34 (m, 4H), 2.99 (t, J=7.9 Hz, 2H),1.77-1.63 (m, 2H), 1.53-1.38 (m, 2H), 0.92 (t, J=7.3 Hz, 3H). ESI-MS(m/z): 300.1 [M+H]⁺.

2-(butylthio)-6-chlorothieno[2,3-b]pyridine. To the solution of2-bromo-6-chlorothieno[2,3-b]pyridine (40 mg, 0.16 mmol) in THF (2 mL)at −78° C. was added n-BuLi (0.32 mmol, 2.0 equiv.; 1.6 M solution inhexanes). The traction mixture was stirred for 5 min. and1,2-dibutyldisulfane (0.48 mmol, 85.4 mg) was then added. The reactionmixture was stirred at −78° C. for additional 1 h, quenched with waterand diluted with EtOAc. The organic layer was separated, dried overMgSO4, filtered and concentrated to give crude product, which waspurified by flash column chromatography (95/5 Hexane/EtOAc) to givedesigned product in 91% isolated yield.

¹H NMR (400 MHz, CDCl3) δ 7.81 (d, J=8.3 Hz, 1H), 7.24 (d, J=8.3 Hz,1H), 7.13 (s, 1H), 3.01-2.89 (m, 2H), 1.74-1.59 (m, 2H), 1.52-1.36 (m,2H), 0.91 (t, J=7.4 Hz, 3H). ESI-MS (m/z): 258.0 [M+H]⁺.

2-(butylthio)-6-chloro-3-nitrothieno[2,3-b]pyridine was prepared in 53%yield according procedure described by Nardine (Meth-Cohn, O.; Narine,B. Tetrahedon Lett. 1978, 23, 2045). ¹H NMR (400 MHz, CDCl₃) δ 8.68 (d,J=8.6 Hz, 1H), 7.45 (d, J=8.6 Hz, 1H), 3.15 (t, J=7.4 Hz, 2H), 1.95-1.73(m, 2H), 1.68-1.41 (m, 2H), 0.99 (t, J=7.4 Hz, 3H). ESI-MS (m/z): 303.0[M+H]⁺.

SW208435 2-(butylthio)-6-phenylthieno[2,3-b]pyridin-3-amine.Phenylboronic acid (37 mg, 0.30 mmol, 2.0 equiv),2-(butylthio)-6-chloro-3-nitrothieno[2,3-b]pyridine (46 mg, 0.15 mmol,1.0 equiv), Cesium Carbonate (0.30 mmol, 2.0 equiv.), PdCl₂dppf (10 mol%), Copper Chloride (0.15 mmol, 15 mg, 1.0 equiv.) were heated in DMF at100° C. for 12 h. After cooling to r.t. the reaction mixture was dilutedwith EtOAc and washed with water and next brine. The organic layer wasdried over magnesium sulfate and the solvent was removed under reducedpressure. The crude product was purified by preparative TLC(AcOEt/Hexanes: 2/8) to afford2-(butylthio)-3-nitro-6-phenylthieno[2,3-b]pyridine. ESI-MS (m/z): 345.1[M+H]⁺. 2-(butylthio)-3-nitro-6-phenylthieno[2,3-b]pyridine (0.017 mmol,6 mg) was dissolved in a mixed solvent of acetic acid (0.12 mL) andconc. hydrochloric acid (one drop). Zinc (13 mg) was added at 0° C.After the mixture was stirred for 30 minutes, the reaction mixture wasfiltered, and the filtrate was neutralized with an aqueous solution ofNaHCO₃, and extracted with DCM. The organic layer was washed with waterand then with a saturated aqueous solution of sodium chloride, and driedover sodium sulfate. Subsequently, the solvent was evaporated to obtaindesigned product. ¹H NMR (400 MHz, CDCl₃) δ 7.65-7.54 (m, 3H), 7.50-7.40(m, 2H), 7.35-7.28 (m, 1H), 7.14 (d, J=8.4 Hz, 1H), 3.35-3.18 (m, 2H),1.80-1.65 (m, 2H), 1.54-1.38 (m, 2H), 0.95 (t, J=7.3 Hz, 3H). ESI-MS(m/z): 315.1 [M+H]⁺.

2-bromo-6-chlorothieno[2,3-b]pyridine was prepared according proceduredescribed by Nardine. ¹¹H NMR (400 MHz, CDCl₃) δ 7.87 (d, J=8.4 Hz, 1H),7.28 (s, 1H), 7.27 (d, J=8.4 Hz, 1H). ESI-MS (m/z): 249 [M+H]⁺.

3-nitro-6-(thiophen-2-yl)pyridin-2-amine. Thiophene boronic acid (742mg, 5.8 mmol, 2.0 equiv), 6-chloro-3-nitropyridin-2-amine (500 mg, 2.9mmol, 1.0 equiv), Cesium Carbonate (5.8 mmol, 2.0 equiv.), PdCl₂dppf (10mol %), Copper Chloride (2.9 mmol, 1.0 equiv.) in DMF were heated at100° C. for 12 h. After cooling to r.t. the reaction mixture was dilutedwith EtOAc and washed with water and next brine. The organic layer wasdried over magnesium sulfate and the solvent was removed under reducedpressure. The crude product was purified by column chromatography(hexanes/EtOAc: 8/2) to afford 3-nitro-6-(thiophen-2-yl)pyridin-2-aminein 63% yield. ¹H NMR (400 MHz CDCl₃) δ 8.42 (d, J=8.7 Hz, 1H), 7.70 (dd,J=3.8, 1.1 Hz, 1H), 7.54 (dd, J=5.0, 1.1 Hz, 1H), 7.15 (dd, J=5.0, 3.8Hz, 1H), 7.09 (d, J=8.7 Hz, 1H). ESI-MS (m/z): 222 [M+H]⁺.

6-(thiophen-2-yl)pyridine-2,3-diamine. The starting material,3-nitro-6-(thiophen-2-yl)pyridin-2-amine (1.20 mmol, 265.4 mg), wasdissolved in a 5:1 acetone/water mixture. Zinc (12.0 mmol, 784 mg, 10eq) and ammonium chloride (18 mmol, 962.5 mg, 15 eq) were added to thesolution, which was stirred at room temperature for 1 hour. The solutionwas then filtered through a celite pad and washed with ethyl acetate.The filtrate was extracted twice with brine then the aqueous layer wasback extracted with EtOAc. The combined organic layers were dried overmagnesium sulfate, filtered, and concentrated under reduced pressure.Further purification by column chromatography gave 118.2 mg of6-(thiophen-2-yl)pyridine-2,3-diamine (52%). ¹H NMR (400 MHz, CD₃OD) δ7.34 (dd, J=3.6, 1.1 Hz, 1H), 7.25 (dd, J=5.1, 1.1 Hz, 1H), 7.00 (dd,J=5.1, 3.6 Hz, 1H), 6.96-6.86 (m, 2H), 4.85 (s, 4H). ESI-MS (m/z): 192[M+H]⁺.

5-(thiophen-2-yl)-1,3-dihydro-2H-imidazo[4,5-b]pyridine-2-thione.Thiourea (16.97 mmol, 223.0 mg, 5 eq) was added to6-(thiophen-2-yl)pyridine-2,3-diamine. The solution was heated at 170°C. for 2 hours. The addition of ethanol room temperature produced solidwhich was filtered to give 112.5 mg of5-(thiophen-2-yl)-1,3-dihydro-2H-imidazo[4,5-b]pyridine-2-thione (82%).¹H NMR (400 MHz, (CD₃)₂SO) δ 7.69 (dd, J=3.7, 1.2 Hz, 1H), 7.66 (d,J=8.3 Hz, 1H), 7.56 (dd, J=5.1, 1.1 Hz, 1H), 7.47 (d, J=8.2 Hz, 1H),7.15-7.09 (m, 1H). ESI-MS (m/z): 235 [M+2H]⁺.

SW208494 2-(butylthio)-5-(thiophen-2-yl)-3H-imidazo[4,5-b]pyridine. Amixture of5-(thiophen-2-yl)-1,3-dihydro-2H-imidazo[4,5-b]pyridine-2-thione (0.39mmol, 92 mg), potassium carbonate (0.45 mmol, 61.9 mg, 1.1 eq),1-bromobutane (0.39 mmol, 42.8 μL, 1 eq), 18-Crown-6 (0.039 mmol, 10.5mg, 0.1 eq), and DMF (2.67 mL) was heated at 80° C. for 3 hours. Thissolution was then diluted with EtOAc and washed with water. The organiclayer was dried over magnesium sulfate, filtered, and concentrated underhigh pressure to give 74.4 mg of SW2084942-(butylthio)-5-(thiophen-2-yl)-3H-imidazo[4,5-b]pyridine (65%). ¹H NMR(400 MHz, CDCl₃) δ 7.95-7.83 (m, 1H), 7.61-7.53 (m, 2H), 7.36 (d, J=5.1,1H), 7.16-7.06 (m, 1H), 3.28 (t, J=7.3 Hz, 2H), 1.76-1.62 (m, 2H), 1.481.32 (m, 2H), 0.90 (t, J=7.4 Hz, 3H). ESI-MS (m/z): 290 [M+H]⁺.

SW208495. 2-(butylsulfinyl)-5-(thiophen-2-yl)-3H-imidazo[4,5-b]pyridine.Chloroform (450 μL), acetic acid (450 μL), and hydrogen peroxide (0.376mmol, 2.0 eq, 40 μL) were added to SW2084942-(butylthio)-5-(thiophen-2-yl)-3H-imidazo[4,5-b]pyridine and heated at45° C. for 2.5 hours. The solution was then diluted with EtOAc andwashed with 10% acetic acid. The organic layer was separated, dried withmagnesium sulfate, filtered, concentrated, and purified to give 16.8 mgof SW208495. ¹H NMR (400 MHz, CDCl₃) δ 8.06 (d, J=8.5 Hz, 1H), 7.83-7.67(m, 1H), 7.68-7.60 (m, 1H), 7.41 (d, J=5.3 Hz, 1H), 7.20-7.05 (m, 1H),3.44-3.17 (m, 2H), 1.89-1.58 (m, 2H), 1.59-1.40 (m, 2H), 0.93 (t, J=7.3Hz, 3H). ESI-MS (m/z): 306 [M+H]⁺.

SW208662.6-(butylsulfinyl)-2-phenyl-4-(piperidin-1-yl)thieno[2,3-d]pyrimidine.Acetic acid (50 μl) and hydrogen peroxide (5.0 μl, 30% solution inwater) were added to the solution of2-(butylthio)-6-phenyl-4-(piperidin-1-yl)thieno[2,3-b]pyrimidine (10 mg,0.026 mmol) in chloroform (50 μl). The reaction mixture was stirred at32° C. for 45 min. Once complete, the reaction was diluted with EtOAcand was washed with saturated NaHCO₃ solution, dried over magnesiumsulfate, filtered and concentrated under reduce pressure to givedesigned product. ¹H NMR (400 MHz, CDCl₃) δ 8.54-8.34 (m, 2H), 7.73 (s,1H), 7.55-7.36 (m, 3H), 4.09-3.86 (m, 4H), 3.24-2.94 (m, 2H), 1.97-1.36(m, 10H), 0.93 (t, J=7.3 Hz, 3H). ESI-MS (m/z): 400.1 [M+H]⁺.

2-(butylthio)-6-phenyl-4-(piperidin-1-yl)thieno[2,3-b]pyrimidine.2-(butylthio)-6-chloro-4-(piperidin-1-yl)thieno[2,3-b]pyrimidine (52 mg,0.15 mmol), phenylboronic acid (27 mg, 0.22 mmol, 1.5 equiv), PotassiumCarbonate (0.3 mmol, 2.0 equiv.), PdCl2dtbpf (10 mol mol %), inCH3CN:H2O (2:1) were heated at 100° C. overnight. After cooling to r.t.the reaction mixture was diluted with EtOAc and washed with water. Theorganic layer was dried over magnesium sulfate and the solvent wasremoved under reduced pressure. The crude product was purified by flashchromatography to afford designed product. 1H NMR (400 MHz, CHCl3) δ8.49-8.36 (m, 2H), 7.51-7.36 (m, 3H), 7.29 (s, 1H), 3.95-3.85 (m, 4H),2.90 (t, J=7.4 Hz, 2H), 1.76-1.73 (m, 6H), 1.70-1.59 (m, 2H), 1.48-1.39(m, 2H), 0.91 (t, J=7.4 Hz, 3H). ESI-MS (m/z): 384.0 [M+H]+.

2-(butylthio)-6-chloro-4-(piperidin-1-yl)thieno[2,3-b]pyrimidine. To thesolution of 6-chloro-4-(piperidin-1-yl)thieno[2,3-b]pyrimidine (52 mg,0.20 mmol) in THF was added n-BuLi (0.4 mmol, 2.0 equiv., 1.6 M solutionin hexanes) at −78° C. The reaction mixture was stirred for 5 min and1,2-dibutyldisulfane (0.80 mmol, 4.0 equiv.) in THF was added. Thereaction mixture was stirred for additional 1 h at −78° C. and thenquenched. The crude product was purified by flash chromatography toafford designed product in 74% yield. ¹H NMR (400 MHz, CHCl₃) δ 7.24 (s,1H), 3.93-3.74 (m, 4H), 2.83 (t, J=7.3 Hz, 2H), 1.82-1.66 (m, 6H),1.66-1.53 (m, 2H), 1.49-1.33 (m, 2H), 0.89 (t, J=7.3 Hz, 3H). ESI-MS(m/z): 342.1 [M+H]⁺.

6-chloro-4-(piperidin-1-yl)thieno[2,3-b]pyrimidine.4,6-dichlorothieno[2,3-b]pyrimidine (50 mg, 0.24 mmol) and piperidine(0.36 mmol, 1.5 equiv.) in EtOH were stirred at room temperatureovernight. The solvent was evaporated and crude compound purified byflash chromatography to give designed product in quantitative yield. ¹HNMR (400 MHz, CDCl₃) δ 7.28 (d, J=6.1 Hz, 1H), 7.18 (d, J=6.2 Hz, 1H),4.01-3.67 (m, 4H), 1.92-1.63 (m, 6H). ESI-MS (m/z): 254.0 [M+H]⁺.

SW208776. 6-(butylsulfinyl)-2,4-diphenylthieno[2,3-d]pyrimidine. Aceticacid (250 μl) and hydrogen peroxide (20 μl, 30% solution in water) wereadded to the solution of6-(butylthio)-2,4-diphenylthieno[2,3-d]pyrimidine (35 mg, 0.1 mmol) inchloroform (250 μl). The reaction mixture was stirred at 32° C. for 45min. Once complete, the reaction was diluted with EtOAc and was washedwith saturated NaHCO₃ solution, dried over magnesium sulfate, filteredand concentrated under reduce pressure to give designed product. ¹H NMR(400 MHz, CDCl₃) δ 8.69-8.59 (m, 2H), 8.09-7.99 (m, 2H), 7.95 (s, 1H),7.65-7.56 (m, 3H), 7.56-7.45 (m, 3H), 3.18-3.02 (m, 2H), 1.87-1.64 (m,2H), 1.54-1.42 (m, 2H), 0.94 (t, J=7.3 Hz, 3H). ESI-MS (m/z): 393.1[M+H]⁺.

6-(butylthio)-2,4-diphenylthieno[2,3-d]pyrimidine. To the solution of2,4-diphenylthieno[2,3-d]pyrimidine (53 mg, 0.28 mmol) in THF was addedn-BuLi (0.56 mmol, 2.0 equiv., 225 μL, 2.5 M solution in hexanes) at−78° C. The reaction mixture was stirred for 5 min and1,2-dibutyldisulfane (1.14 mmol, 4.0 equiv.) in THF was added. Thereaction mixture was stirred for additional 1 h at −78° C. and thenquenched. The crude product was purified by flash chromatography toafford designed product. ¹H NMR (400 MHz, CDCl₃) δ 8.62-8.56 (m, 2H),8.06-7.98 (m, 2H), 7.61-7.41 (m, 7H), 3.01 (t, J=7.3, 2H), 1.76-1.62 (m,2H), 1.55-1.38 (m, 2H), 0.92 (t, J=7.4 Hz, 3H). ESI-MS (m/z): 377.1[M+H]⁺.

2,4-diphenylthieno[2,3-d]pyrimidine. 2,4-dichlorothieno[2,3-d]pyrimidine(100 mg, 0.50 mmol), phenylboronic acid (242 mg, 2.0 mmol, 4.0 equiv),Potassium Carbonate (1.5 mmol, 3.0 equiv.), Pd(OAc)₂ (5 mol mol %),SPhos (10 mol %) in CH₃CN:H₂O (1.5:1) were heated at 100° C. overnight.After cooling to r.t. the reaction mixture was diluted with EtOAc andwashed with water. The organic layer was dried over magnesium sulfateand the solvent was removed under reduced pressure. The crude productwas purified by flash chromatography to afford designed product. ¹H NMR(400 MHz, CDCl₃) δ 8.70-8.59 (m, 2H), 8.14-8.02 (m, 2H), 7.65-7.44 (m,8H). ESI-MS (m/z): 289.0 [M+H]⁺.

SW208777.2-(butyl(λ¹-oxidanyl)-λ³-sulfanyl)-4-(pyridin-3-yl)-6-(thiazol-2-yl)thieno[2,3-b]pyridin-3-aminewas prepared using synthetic procedures described for the preparation ofanalog SW033291. ¹H NMR (400 MHz, CDCl₃) δ 8.80 (s, 1H), 8.78 (dd,J=4.9, 1.7 Hz, 1H), 8.04 (s, 1H), 7.91 (d, J=3.2 Hz, 1H), 7.86 (d, J=6.4Hz, 1H), 7.51 (d, J=3.1 Hz, 1H), 7.47 (dd, J=7.8, 4.8 Hz, 1H), 4.53 (s,2H), 3.28 (ddd, J=12.8, 8.8, 6.3 Hz, 1H), 3.11 (ddd, J=12.8, 8.9, 6.9Hz, 1H), 1.86-1.70 (m, 2H), 1.57-1.38 (m, 2H), 0.94 (t, J=7.3 Hz, 3H).ESI-MS (m/z): 415.0 [M+H]⁺.

SW208780.2-(isopropyl(λ¹-oxidanyl)-λ³-sulfanyl)-4-(pyridin-3-yl)-6-(thiazol-2-yl)thieno[2,3-b]pyridin-3-aminewas prepared using synthetic procedures described for the preparation ofanalog SW033291. ¹H NMR (400 MHz, CDCl₃) δ 8.87-8.70 (m, 2H), 8.05 (s,1H), 7.92 (d, J=3.1 Hz, 1H), 7.85 (dd, J=7.8, 2.4 Hz, 1H), 7.51 (d,J=3.2 Hz, 1H), 7.47 (dd, J=7.9, 4.9 Hz, 1H), 4.57 (s, 2H), 3.38 (p,J=6.8 Hz, 1H), 1.43 (d, J=6.8 Hz, 3H), 1.29 (d, J=6.8 Hz, 3H). ESI-MS(m/z): 400.1 [M+H]⁺.

SW209123. Ethyl3-amino-4-(4-bromophenyl)-2-(butylsulfinyl)-6-(thiazol-2-yl)thieno[2,3-b]pyridine-5-carboxylatewas prepared using synthetic procedures described for the preparation ofanalog SW033291. ¹H NMR (400 MHz, CDCl₃) δ 7.86 (d, J=3.2 Hz, 1H),7.69-7.60 (m, 2H), 7.48 (d, J=3.2 Hz, 1H), 7.36-7.27 (m, 2H), 4.12 (q,J=7.2 Hz, 2H), 3.26 (ddd, J=12.9, 8.8, 6.3 Hz, 1H), 3.08 (ddd, J=12.9,8.8, 6.3 Hz, 1H), 1.80-1.63 (m, 2H), 1.58-1.37 (m, 2H), 1.06 (t, J=7.2Hz, 3H), 0.93 (t, J=7.3 Hz, 3H). ESI-MS (m/z): 564.0 [M+H]⁺.

SW209124.2-(butylsulfinyl)-4,6-di(thiazol-2-yl)thieno[2,3-b]pyridin-3-amine wasprepared using synthetic procedures described for the preparation ofanalog SW033291. ¹H NMR (400 MHz, CDCl₃) δ 8.48 (s, 1H), 8.01 (d, J=3.1Hz, 1H), 7.96 (d, J=3.2 Hz, 1H), 7.61 (d, J=3.3 Hz, 1H), 7.52 (d, J=3.1Hz, 1H), 6.69 (s, 2H), 3.30 (ddd, J=12.8, 9.2, 6.0 Hz, 1H), 3.14 (ddd,J=12.8, 9.2, 6.4 Hz, 1H), 1.83-1.60 (m, 2H), 1.43-1.53 (m, 2H), 0.93 (t,J=7.3 Hz, 3H). ESI-MS (m/z): 421.0 [M+H]⁺.

SW209125.2-(butylsulfinyl)-4-(1-methyl-1H-imidazol-2-yl)-6-(thiazol-2-yl)thieno[2,3-b]pyridin-3-aminewas prepared using synthetic procedures described for the preparation ofanalog SW033291. ¹H NMR (400 MHz, CDCl₃) δ 8.13 (s, 1H), 7.91 (d, J=3.2Hz, 1H), 7.50 (d, J=3.1 Hz, 1H), 7.24 (d, J=1.2 Hz, 1H), 7.13 (d, J=1.2Hz, 1H), 5.78 (s, 2H), 3.80 (s, 3H), 3.26 (ddd, J=12.8, 9.1, 6.0 Hz,1H), 3.10 (ddd, J=12.8, 9.2, 6.5 Hz, 1H), 1.82-1.57 (m, 2H), 1.56-1.35(m, 2H), 0.92 (t, J=7.3 Hz, 3H). ESI-MS (m/z): 418.1 [M+H]⁺.

SW209126.2-(butylsulfinyl)-6-(1-methyl-1H-imidazol-2-yl)-4-phenylthieno[2,3-b]pyridin-3-aminewas prepared using synthetic procedures described for the preparation ofanalog SW033291. ¹H NMR (400 MHz, CDCl₃) δ 8.08 (s, 1H), 7.58-7.32 (m,5H), 7.11 (d, J=1.1 Hz, 1H), 7.00 (d, J=1.1 Hz, 1H), 4.58 (s, 2H), 4.19(s, 3H), 3.27 (ddd, J=12.7, 9.0, 6.0 Hz, 1H), 3.08 (ddd, J=12.8, 9.1,6.6 Hz, 1H), 1.79-1.60 (m, 2H), 1.56-1.37 (m, 2H), 0.92 (t, J=7.3 Hz,3H). ESI-MS (m/z): 411.1 [M+H]⁺.

SW209277.6-(butylsulfinyl)-2-(1-methyl-1H-imidazol-2-yl)-4-phenylthieno[2,3-d]pyrimidin-5-aminewas prepared using synthetic procedures described for the preparation ofanalog SW208065. ¹H NMR (400 MHz, CDCl₃) δ 7.71 (dd, J=6.9, 2.8 Hz, 2H),7.63-7.49 (m, 3H), 7.29 (s, 1H), 7.07 (s, 1H), 4.85 (s, 2H), 4.18 (s,3H), 3.29 (ddd, J=12.8, 8.6, 6.3 Hz, 1H), 3.11 (ddd, J=12.8, 8.7, 6.9Hz, 1H), 1.83-1.65 (m, 2H), 1.59-1.39 (m, 2H), 0.94 (t, J=7.3 Hz, 3H).ESI-MS (m/z): 412.1 [M+H]⁺.

SW209278.6-(butylsulfinyl)-2-(oxazol-4-yl)-4-phenylthieno[2,3-d]pyrimidin-5-aminewas prepared using synthetic procedures described for the preparation ofanalog SW208065. ¹H NMR (400 MHz, CDCl₃) δ 8.51 (d, J=1.1 Hz, 1H), 8.01(d, J=1.1 Hz, 1H), 7.75-7.61 (m, 2H), 7.62-7.48 (m, 3H), 4.56 (s, 2H),3.29 (ddd, J=12.9, 8.8, 6.3 Hz, 1H), 3.09 (ddd, J=12.9, 8.9, 6.9 Hz,1H), 1.81-1.64 (m, 2H), 1.56-1.39 (m, 2H), 0.93 (t, J=7.3 Hz, 3H).ESI-MS (m/z): 399.1 [M+H]⁺.

SW209279.2-(isopropylsulfinyl)-4-(1-methyl-1H-imidazol-2-yl)-6-(thiazol-2-yl)thieno[2,3-b]pyridin-3-aminewas prepared using synthetic procedures described for the preparation ofanalog SW033291. ¹H NMR (400 MHz, CDCl₃) δ 8.14 (s, 1H), 7.92 (d, J=3.2Hz, 1H), 7.51 (d, J=3.2 Hz, 1H), 7.24 (s, 1H), 7.13 (d, J=1.2 Hz, 1H),5.92 (s, 2H), 3.80 (s, 3H), 3.38 (p, J=6.8 Hz, 1H), 1.44 (d, J=6.8 Hz,3H), 1.25 (d, J=6.8 Hz, 3H). ESI-MS (m/z): 404.1 [M+H]⁺.

SW209280.4-(1-methyl-1H-imidazol-2-yl)-2-(propylsulfinyl)-6-(thiazol-2-yl)thieno[2,3-b]pyridin-3-aminewas prepared using synthetic procedures described for the preparation ofanalog SW033291. ¹H NMR (400 MHz, CDCl₃) δ 8.14 (s, 1H), 7.92 (d, J=3.2Hz, 1H), 7.51 (d, J=3.1, 1H), 7.25 (d, J=1.3 Hz, 1H), 7.14 (d, J=1.2 Hz,1H), 5.97 (s, 2H), 3.80 (s, 3H), 3.27 (ddd, J=12.7, 8.3, 6.5 Hz, 1H),3.07 (ddd, J=12.8, 8.4, 7.1 Hz, 1H), 1.85-1.69 (m, 2H), 1.07 (t, J=7.4Hz, 3H). ESI-MS (m/z): 404.1 [M+H]⁺.

SW209415.2-(butylsulfinyl)-4-(1,2-dimethyl-1H-imidazol-5-yl)-6-(thiazol-2-yl)thieno[2,3-b]pyridin-3-amine.To the solution of2-(((butylsulfinyl)methyl)thio)-4-(1,2-dimethyl-1H-imidazol-5-yl)-6-(thiazol-2-yl)nicotinonitrile(0.14 mmol, 60 mg) in DMF (600 μl)/MeOH (300 μl) was added KOH (0.084mmol, 4.70 mg, 0.6 equiv., 2.0 M in water). The reaction mixture wasstirred at 32° C. for 20 min. Once complete, the reaction was dilutedwith EtOAc and acidified to pH 7 with 5% aq. solution of AcOH, theorganic phase was separated and aqueous layer was extracted twice withEtOAc, dried over magnesium sulfate, filtered and concentrated underreduced pressure. The crude product was purified by flash chromatographyto afford designed product in 97% isolated yield. ¹H NMR (400 MHz,CDCl₃) δ 8.03 (s, 1H), 7.90 (d, J=3.1 Hz, 1H), 7.50 (d, J=3.2 Hz, 1H),7.11 (s, 1H), 4.76 (s, 2H), 3.39 (s, 3H), 3.27 (ddd, J=12.9, 8.7, 6.4Hz, 1H), 3.09 (ddd, J=12.8, 8.8, 6.9 Hz, 1H), 2.47 (s, 3H), 1.83-1.62(m, 2H), 1.57-1.38 (m, 2H), 0.93 (t, J=7.3 Hz, 3H). ESI-MS (m/z): 432.1[M+H]⁺. Two enantiomers of SW209415 can be separated by chiral HPLC:Chiralpak AD-H, 10×250 mm, 5 μM, 100% MeOH.

2-(((butylsulfinyl)methyl)thio)-4-(1,2-dimethyl-1H-imidazol-5-yl)-6-(thiazol-2-yl)nicotinonitrile.To the solution of2-(((butylthio)methyl)thio)-4-(1,2-dimethyl-1H-imidazol-5-yl)-6-(thiazol-2-yl)nicotinonitrile(85 mg, 0.205 mmol) in CHCl₃/AcOH (1:1, 0.15 M) was added H₂O₂ (0.31mmol, 1.5 equiv. 30% solution in water). The reaction mixture wasstirred at 32° C. for 40 min. Once complete, the reaction was dilutedwith EtOAc and was washed with saturated NaHCO₃ solution, dried overmagnesium sulfate, filtered and concentrated under reduce pressure togive designed product in 92% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.98 (d,J=3.1 Hz, 1H), 7.94 (s, 1H), 7.60 (d, J=3.1 Hz, 1H), 7.43 (s, 1H), 4.72(d, J=13.1 Hz, 1H), 4.41 (d, J=13.1 Hz, 1H), 3.63 (s, 3H), 2.96 (dt,J=12.9, 8.2 Hz, 1H), 2.84 (dt, J=12.9, 7.5 Hz, 1H), 2.51 (s, 3H),1.94-1.74 (m, 2H), 1.63-1.38 (m, 2H), 0.95 (t, J=7.4 Hz, 3H). ESI-MS(m/z): 432.1 [M+H]⁺.

2-(((butylthio)methyl)thio)-4-(1,2-dimethyl-1H-imidazol-5-yl)-6-(thiazol-2-yl)nicotinonitrile.To a suspension of3-(1,2-dimethyl-1H-imidazol-5-yl)-1-(thiazol-2-yl)prop-2-en-1-one (0.31mmol, 72 mg) and 2-cyanothioacetamide (0.93 mmol, 93 mg, 3.0 equiv.) inEtOH (1.5 mL), a few drops of piperidine were added. After being stirredat 80° C. for 2 h, EtOH was evaporated and crude product was redissolvedin CH₃CN. Butyl(chloromethyl)sulfane (0.62 mmol, 85.5 mg) and Et₃N (0.93mmol, 94.1 mg, 130 μL) were then added and the reaction mixture wasstirred at 80° C. for 20 min. Once complete, the reaction was dilutedwith EtOAc and water. The organic phase was separated and aqueous layerwas extracted twice with EtOAc. The combined extractions were washedwith saturated NaCl solution, dried over magnesium sulfate, filtered andconcentrated under reduced pressure. The residue was purified by flashchromatography to give 99 mg of designed product (77%). ¹H NMR (400 MHz,CDCl₃) δ 7.96 (d, J=3.1 Hz, 1H), 7.85 (s, 1H), 7.56 (d, J=3.1 Hz, 1H),7.37 (s, 1H), 4.49 (s, 2H), 3.60 (s, 3H), 2.72 (t, J=7.4 Hz, 2H), 2.48(s, 3H), 1.62 (p, J=7.3 Hz, 2H), 1.40 (h, J=7.3 Hz, 2H), 0.90 (t, J=7.3Hz, 3H). ESI-MS (m/z): 416.6 [M+H]⁺.

(E)-3-(1,2-dimethyl-1H-imidazol-5-yl)-1-(thiazol-2-yl)prop-2-en-1-one.To a solution of 1,5-dimethyl-1H-imidazole-2-carbaldehyde (2.0 mmol, 250mg) in 6 ml of CH₃CN was added1-(thiazol-2-yl)-2-(triphenyl-15-phosphanylidene)ethan-1-one (4.0 mmol,1.55 g, 2.0 equiv.). The reaction mixture was stirred at 90° C. for 48h. Once complete, solvent was evaporated and residue was purified byflash chromatography to give 331 mg of designed product (71%). 1H NMR(400 MHz, Methanol-d4) δ 8.08 (d, J=3.0 Hz, 1H), 7.97 (d, J=3.0 Hz, 1H),7.90 (d, J=15.9 Hz, 1H), 7.76 (d, J=15.9 Hz, 1H), 7.60 (s, 1H), 3.72 (s,3H), 2.43 (s, 3H). ESI-MS (m/z): 234.3 [M+H]⁺.

SW209428.2-(butylsulfinyl)-4-(2-methyl-1H-imidazol-5-yl)-6-(thiazol-2-yl)thieno[2,3-b]pyridin-3-aminewas prepared using synthetic procedures described for the preparation ofanalog SW209415. ¹H NMR (400 MHz, CDCl₃) δ 10.51 (s, 1H), 8.10 (s, 1H),7.89 (d, J=3.2 Hz, 1H), 7.46 (d, J=3.2 Hz, 1H), 7.40 (s, 1H), 3.31 (ddd,J=12.8, 9.3, 5.8 Hz, 1H), 3.15 (ddd, J=12.8, 9.3, 6.2 Hz, 1H), 2.42 (s,3H), 1.79-1.58 (m, 2H), 1.57-1.38 (m, 2H), 0.93 (t, J=7.3 Hz, 3H).ESI-MS (m/z): 418.1 [M+H]⁺.

SW211688. 4-(1,2-dimethyl-1H-imidazol-5-yl)-2-((3-methoxypropyl)sulfinyl)-6-(thiazol-2-yl)thieno[2,3-b]pyridin-3-amine was preparedusing synthetic procedures described for the preparation of analogSW209415. ¹H NMR (400 MHz, Acetone-d6) δ 8.03 (s, 1H), 7.99 (d, J=3.2Hz, 1H), 7.82 (d, J=3.2 Hz, 1H), 7.09 (s, 1H), 5.06 (s, 2H), 3.51 (s,3H), 3.48 (t, J=6.1 Hz, 2H), 3.26 (s, 3H), 3.26-3.18 (m, 1H), 3.18-3.12(m, 1H), 2.43 (s, 3H), 2.00-1.89 (m, 2H). ESI-MS (m/z): 448.1 [M+H]⁺.

SW211689. 4-(1,2-dimethyl-1H-imidazol-5-yl)-2-((2-methoxyethyl)sulfinyl)-6-(thiazol-2-yl)thieno[2,3-b]pyridin-3-amine was preparedusing synthetic procedures described for the preparation of analogSW209415. ¹H NMR (400 MHz, CDCl₃) δ 8.05 (s, 1H), 7.92 (d, J=3.2 Hz,1H), 7.51 (d, J=3.2 Hz, 1H), 7.11 (s, 1H), 4.73 (s, 2H), 3.88-3.82 (m,1H), 3.75-3.62 (m, 1H), 3.57 (ddd, J=13.1, 6.0, 3.9 Hz, 1H), 3.40 (s,3H), 3.37 (s, 3H), 3.25 (ddd, J=12.8, 8.0, 4.4 Hz, 1H), 2.48 (s, 3H).ESI-MS (m/z): 434.1 [M+H]⁺.

SW212344.2-(butylsulfinyl)-4-(2-isopropyl-1-methyl-1H-imidazol-5-yl)-6-(thiazol-2-yl)thieno[2,3-b]pyridin-3-aminewas prepared using synthetic procedures described for the preparation ofanalog SW209415. ¹H NMR (400 MHz, CDCl₃) δ 8.06 (s, 1H), 7.92 (d, J=3.1Hz, 1H), 7.51 (d, J=3.2 Hz, 1H), 7.15 (s, 1H), 4.71 (s, 2H), 3.41 (s,3H), 3.27 (ddd, J=13.0, 8.5, 6.5 Hz, 1H), 3.19-2.98 (m, 2H), 1.83-1.59(m, 2H), 1.58-1.41 (m, 2H), 1.39 (d, J=6.7 Hz, 6H), 0.94 (t, J=7.3 Hz,3H). ESI-MS (m/z): 460.1 [M+H]⁺.

SW212345.2-(butylsulfinyl)-4-(2-cyclopropyl-1-methyl-1H-imidazol-5-yl)-6-(thiazol-2-yl)thieno[2,3-b]pyridin-3-aminewas prepared using synthetic procedures described for the preparation ofanalog SW209415. ¹H NMR (400 MHz, CDCl₃) δ 8.04 (s, 1H), 7.91 (d, J=3.1Hz, 1H), 7.50 (d, J=3.1 Hz, 1H), 7.07 (s, 1H), 4.77 (s, 2H), 3.51 (s,3H), 3.27 (ddd, J=12.9, 8.7, 6.4 Hz, 1H), 3.10 (ddd, J=12.9, 8.8, 6.9Hz, 1H), 1.95-1.78 (m, 1H), 1.81-1.62 (m, 2H), 1.58-1.37 (m, 2H),1.17-0.98 (m, 4H), 0.93 (t, J=7.3 Hz, 3H). ESI-MS (m/z): 458.1 [M+H]⁺.

2-bromo-1-(thiazol-2-yl)ethan-1-one. n-Butyllithium (24.7 mL, 0.0617mol, 2.5M in Hexane) was added dropwise to a solution of 2-thiazole (5.0g, 0.059 mol) in anhydrous diethyl ether (48.8 mL) at −78° C. After 15minutes, ethylbromoacetate (6.84 mL, 0.0617 mol) was added, the coldbath was removed and the solution was allowed to warm to roomtemperature. The reaction mixture was diluted with ether and water. Theorganic layer was separated, dried over Na2SO4, filtered andconcentrated under reduced pressure. The crude product was suspended inhexanes and heated to reflux for 15 minutes then the product wasdecanted off leaving the impure oil. This was repeated 5 times to give awhite solid with 88% yield. ¹H NMR (400 MHz, CDCl3) δ 8.05 (d, J=3.0 Hz,1H), 7.77 (d, J=3.0 Hz, 1H), 4.71 (s, 2H). ESI-MS (m/z): 207.8 [M+H]+.

1-(thiazol-2-yl)-2-(triphenyl-15-phosphanylidene)ethan-1-one. To asolution of 2-bromo-1-(thiazol-2-yl)ethan-1-one (10.7 g, 0.0517 mol) intoluene (337.7 mL), triphenylphosphine (14.1 g, 0.0539 mol) was addedportion wise. The mixture was stirred at room temperature for 3 hours.The yellowish precipitate was removed by filtration, and was washedseveral times with toluene and then petroleum ether. Water was added tothe precipitate and was treated dropwise with 1N NaOH to pH 10 (at pH 7there was a color change from yellow to orange). The mixture was stirredfor 30 minutes at room temperature. The precipitate was removed byfiltration and washed several times with water. The resulting orangesolid, was heated at 50° C. under vacuum to remove any water, giving a96% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.82 (d, J=3.1 Hz, 1H), 7.72 (ddd,J=12.8, 8.3, 1.4 Hz, 6H), 7.61-7.54 (m, 3H), 7.51-7.45 (m, 6H), 7.38(dd, J=3.1, 1.3 Hz, 1H), 5.00 (d, J=23.3 Hz, 1H). ESI-MS (m/z): 387.9[M+H]⁺.

Methyl (E)-4-(3-oxo-3-(thiazol-2-yl)prop-1-en-1-yl)benzoate. In a driedflask, 1-(thiazol-2-yl)-2-(triphenyl-15-phosphanylidene)ethan-1-one (1.5g, 3.9 mmol) and methyl 4-formyl benzoate (634 mg, 3.86 mmol) weredissolved in anhydrous chloroform (19.3 mL) and the solution stirred at71° C. overnight. The solvent was evaporated under reduced pressure andthe solid precipitate was purified using automated flash chromatography(100% DCM) to give a white solid in 76% yield. 1H NMR (400 MHz, CDC3) δ8.10-8.05 (m, 3H), 8.01 (d, J=1.3 Hz, 2H), 7.76 (d, J=8.4 Hz, 2H), 7.72(d, J=3.0 Hz, 1H), 3.93 (s, 3H). ESI-MS (m/z): 274.0 [M+H]+.

Methyl4-(2-(((butylthio)methyl)thio)-3-cyano-6-(thiazol-2-yl)pyridin-4-yl)benzoate.2-cyanothioacetamide (274.8 mg, 2.744 mmol) and methyl(E)-4-(3-oxo-3-(thiazol-2-yl)prop-1-en-1-yl)benzoate (250.0 mg, 0.9147mmol) were combined in a vial that was evacuated and backfilled with 02then ethanol (2.75 mL) and piperdine (2 drops) were added. The solutionwas sparged for a few minutes then stirred at 80° C. for 4 hours. Oncecooled, the solution was filtered, and the precipitate was rinsed withethanol, and then washed in minimal amounts of acetic acid by heating at80° C. for 45 minutes. When cooled, the washed solution was filteredleaving the crude brown/red solid product, which was carried forward tothe next step. Standard alkylation procedure: Butyl(chloromethyl)sulfane(111.2 mg, 0.8059 mmol) in acetonitrile (1.32 mL), was added to theproduct from the first step, and Et3N (168.6 μL, 1.209 mmol) was addedlast. The solution was stirred at 80° C. for 20 minutes. The reactionmixture was diluted with EtOAc and washed with H2O, dried over Na2SO4,filtered, and concentrated under reduced pressure. The crude solid waspurified using automated flash chromatography (80% hexane, 20% EtOAc).This produced a solid in 24% yield. ¹H NMR (400 MHz, CDCl3) δ 8.18 (d,J=8.4 Hz, 2H), 8.02 (s, 1H), 7.98 (d, J=3.1 Hz, 1H), 7.71 (d, J=8.4 Hz,2H), 7.58 (d, J=3.2 Hz, 1H), 4.52 (s, 2H), 3.95 (s, 3H), 2.76 (t, J=7.3Hz, 2H), 1.64 (tt, J=7.7, 6.3 Hz, 2H), 1.42 (h, J=7.3 Hz, 2H), 0.91 (t,J=7.3 Hz, 3H). ESI-MS (m/z): 456.1 [M+H]+.

2-(((butylthio)methyl)thio)-4-(4-(hydroxymethyl)phenyl)-6-(thiazol-2-yl)nicotinonitrile.To the solution of methyl4-(2-(((butylthio)methyl)thio)-3-cyano-6-(thiazol-2-yl)pyridin-4-yl)benzoate(336 mg, 0.737 mmol) in THF (8.41 mL) LiBH₄ (96.3 mg, 4.42 mmol) wasadded at 0° C. The reaction was stirred at room temperature for 36hours, and the reaction was monitored by LC/MS. The reaction mixture wasdiluted with EtOAc and H₂O. The organic layer was dried over Na₂SO₄,filtered, and concentrated under reduced pressure, to give product in96% yield. ¹H NMR (400 MHz, CDCl₃) δ 8.02 (s, 1H), 7.98 (d, J=3.1 Hz,1H), 7.69-7.62 (m, 2H), 7.56 (d, J=3.1 Hz, 1H), 7.56-7.49 (m, 2H), 4.79(d, J=4.3 Hz, 2H), 4.52 (s, 2H), 2.82-2.60 (m, 2H), 1.71-1.58 (m, 2H),1.49-1.33 (m, 2H), 0.91 (t, J=7.4 Hz, 3H). ESI-MS (m/z): 428.1 [M+H]⁺.

Standard oxidation procedure:2-(((butyl(l1-oxidanyl)-l3-sulfanyl)methyl)thio)-4-(4-(hydroxymethyl)phenyl)-6-(thiazol-2-yl)nicotinonitrile.Chloroform (2.53 mL), acetic acid (1.39 mL), and hydrogen peroxide(108.0 μL, 1.057 mmol, 30% solution in water) were added to2-(((butylthio)methyl)thio)-4-(4-(hydroxymethyl)phenyl)-6-(thiazol-2-yl)nicotinonitrile.The solution was stirred at 32° C. for 45 minutes. The reaction mixturewas then diluted with EtOAc and washed with saturated NaHCO3, and theorganic layer was dried over Na2SO4, filtered, and concentrated underreduced pressure to give the desired product in 94% yield. ¹H NMR (400MHz, CDCl3) δ 8.03 (s, 1H), 7.93 (d, J=3.1 Hz, 1H), 7.59 (d, J=8.2 Hz,2H), 7.55 (d, J=3.1 Hz, 1H), 7.48 (d, J=7.9 Hz, 2H), 4.73 (s, 2H), 4.66(d, J=13.1 Hz, 1H), 4.38 (d, J=13.1 Hz, 1H), 2.93 (dt, J=13.0, 8.1 Hz,1H), 2.79 (dt, J=13.0, 7.2 Hz, 1H), 1.84-1.72 (m, 2H), 1.55-1.33 (m,2H), 0.91 (t, J=7.3 Hz, 3H)). ESI-MS (m/z): 444.1 [M+H]+.

SW209510(4-(3-amino-2-(butyl(l1-oxidanyl)-l3-sulfanyl)-6-(thiazol-2-yl)thieno[2,3-b]pyridin-4-yl)phenyl)methanol.t-BuOK (22.78 mg, 0.2028 mmol) was added to2-(((butyl(l1-oxidanyl)-l3-sulfanyl)methyl)thio)-4-(4-(hydroxymethyl)phenyl)-6-(thiazol-2-yl)nicotinonitrile(150 mg, 0.338 mmol) and the vial was evacuated backfilled with N₂ threetimes before adding DMF (1.3 mL). The solution was sparged with N₂ for afew minutes before heating at 32° C. The reaction mixture was monitoredevery five minutes by TLC (80% EtOAc. 20% hexanes) and upon completionwas diluted with EtOAc and washed with 10% AcOH. The organic layer wasthen dried over Na₂SO₄, filtered, and concentrated under reducedpressure. The product was purified using automated flash chromatographyto give an isolated green solid/oil in 16% yield. ¹H NMR (400 MHz,CDCl₃) δ 8.02 (s, 1H), 7.90 (d, J=3.2 Hz, 1H), 7.59-7.40 (m, 5H), 4.80(s, 2H), 4.63 (s, 2H), 3.27 (ddd, J=12.8, 9.0, 6.1 Hz, 1H), 3.10 (ddd,J=12.8, 9.1, 6.6 Hz, 1H), 1.78-1.61 (m, 2H), 1.55-1.40 (m, 2H), 0.93 (t,J=7.3 Hz, 3H). ESI-MS (m/z): 444.1 [M+H]⁺.

SW2095114-(3-amino-2-(butyl(l1-oxidanyl)-l3-sulfanyl)-6-(thiazol-2-yl)thieno[2,3-b]pyridin-4-yl)benzylacetate. This compound was formed during the workup of SW209510 in EtOAc(47% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.99 (s, 1H), 7.87 (d, J=3.2 Hz,1H), 7.56-7.40 (m, 5H), 5.18 (s, 2H), 4.62 (s, 2H), 3.26 (ddd, J=12.8,9.0, 6.1 Hz, 1H), 3.08 (ddd, J=12.8, 9.1, 6.6 Hz, 1H), 2.14 (s, 3H),1.77-1.59 (m, 2H), 1.53-1.37 (m, 2H), 0.92 (t, J=7.3 Hz, 3H). ESI-MS(m/z): 486.1 [M+H]⁺.

4-(3-amino-2-(butyl(l1-oxidanyl)-l3-sulfanyl)-6-(thiazol-2-yl)thieno[2,3-b]pyridin-4-yl)benzaldehyde.MnO₂ (111.3 mg, 1.28 mmol) was added to a solution of SW209510 (56.8 mg,0.128 mmol) in DCM (2.3 mL) and stirred at room temperature overnight.LC/MS indicated that the reaction was incomplete. The reaction wasfiltered over celite, washed with DCM and the filtrate was concentratedunder reduced pressure. The crude mixture was redissolved in DCM (2.3mL) and MnO₂ (5 eq) was added. The solution was left to stir 24 hours atroom temperature, was filtered over celite and washed with DCM. Thefiltrate was concentrated under reduced pressure and the resulting crudeproduct was purified using automated flash chromatography (55% EtOAc,45% hexanes) resulting in 24% isolated yield. ¹H NMR (400 MHz, CDCl₃) δ10.13 (s, 1H), 8.11-7.99 (m, 3H), 7.92 (d, J=3.1 Hz, 1H), 7.75-7.62 (m,2H), 7.51 (d, J=3.2 Hz, 1H), 4.56 (s, 2H), 3.29 (ddd, J=12.8, 8.8, 6.3Hz, 1H), 3.11 (ddd, J=12.8, 8.9, 6.9 Hz, 1H), 1.82-1.66 (m, 2H),1.54-1.41 (m, 2H), 0.94 (t, J=7.3 Hz, 3H). ESI-MS (m/z): 442.1 [M+H]⁺.

SW209513.2-(butyl(l1-oxidanyl)-l3-sulfanyl)-4-(4-((dimethylamino)methyl)phenyl)-6-(thiazol-2-yl)thieno[2,3-b]pyridin-3-amine.To a solution of4-(3-amino-2-(butyl(l1-oxidanyl)-l3-sulfanyl)-6-(thiazol-2-yl)thieno[2,3-b]pyridin-4-yl)benzaldehyde(13.3 mg, 0.0301) in methanol (802.7 μL), dimethylamine (174 μL. 0.301mmol, 2.0M in THF) and acetic acid (1.72 μL, 0.0301 mmol) were added andthe reaction was stirred at room temperature for 90 minutes. Thereaction was then cooled down to 0° C. and sodium cyanoborohydride (3.7mg, 0.060 mmol) was added and the reaction stirred for 2 hours at thistemperature before allowing to warm up to room temperature. After 24hours, more sodium cyanoborohydride (2 eq) was added at 0° C. and leftto stir at room temperature another 24 hours. Nitrogen was used toevaporate the solvent, giving a solid that was diluted with EtOAc andwashed with saturated NaHCO₃. The organic layer was dried over Na₂SO₄,filtered, and concentrated under reduced pressure. The crude product waspurified using flash chromatography (7% MeOH, 93% DCM) isolating theproduct in 13% yield. ¹H NMR (400 MHz, CDCl₃) δ 8.07 (s, 1H), 7.93 (d,J=3.2 Hz, 1H), 7.51 (d, J=3.2 Hz, 1H), 7.49-7.41 (m, 4H), 4.67 (s, 2H),3.55 (s, 2H), 3.36-3.25 (m, 1H), 3.13 (ddd, J=12.8, 9.0, 6.7 Hz, 1H),2.30 (s, 6H), 1.78-1.68 (m, 2H), 1.55-1.44 (m, 2H), 0.95 (t, J=7.3 Hz,3H). ESI-MS (m/z): 471.2 [M+H]⁺.

Methyl (E)-3-(3-oxo-3-(thiazol-2-yl)prop-1-en-1-yl)benzoate. Followedprocedure for methyl(E)-4-(3-oxo-3-(thiazol-2-yl)prop-1-en-1-yl)benzoate using methyl3-formyl benzoate as the starting material. Purified the crude productusing automated flash chromatography (50% EtOAc, 50% hexanes) isolatingthe product in 51% yield. ¹H NMR (400 MHz, CDCl₃) δ 8.41-8.35 (m, 1H),8.11-8.05 (m, 2H), 8.02 (d, J=1.3 Hz, 2H), 7.89-7.83 (m, 1H), 7.72 (d,J=3.0 Hz, 1H), 7.50 (t, J=7.8 Hz, 1H), 3.95 (s, 3H). ESI-MS (m/z): 274.1

Methyl3-(2-(((butylthio)methyl)thio)-3-cyano-6-(thiazol-2-yl)pyridin-4-yl)benzoate.Followed the procedure for methyl4-(2-(((butylthio)methyl)thio)-3-cyano-6-(thiazol-2-yl)pyridin-4-yl)benzoateusing methyl (E)-3-(3-oxo-3-(thiazol-2-yl)prop-1-en-1-yl)benzoate as thestarting material. Isolated product in 87% yield. ¹H NMR (400 MHz,CDCl₃) δ 8.32-8.26 (m, 1H), 8.20 (dt, J=7.9, 1.3 Hz, 1H), 8.04 (s, 1H),7.99 (d, J=3.1 Hz, 1H), 7.85 (ddd, J=7.7, 2.0, 1.1 Hz, 1H), 7.62 (td,J=7.8, 0.6 Hz, 1H), 7.58 (d, J=3.1 Hz, 1H), 4.53 (s, 2H), 3.95 (s, 3H),2.76 (t, J=7.3 Hz, 2H), 1.71-1.59 (m, 2H), 1.49-1.36 (m, 2H), 0.91 (t,J=7.3 Hz, 3H). ESI-MS (m/z): 456.1 [M+Z]⁺.

2-(((butylthio)methyl)thio)-4-(3-(hydroxymethyl)phenyl)-6-(thiazol-2-yl)nicotinonitrile.Followed procedure for2-(((butylthio)methyl)thio)-4-(4-(hydroxymethyl)phenyl)-6-(thiazol-2-yl)nicotinonitrileusing methyl3-(2-(((butylthio)methyl)thio)-3-cyano-6-(thiazol-2-yl)pyridin-4-yl)benzoateas the starting material. Isolated product in 84% yield. ¹H NMR (400MHz, CDCl₃) δ 8.00 (s, 1H), 7.95 (d, J=3.1 Hz, 1H), 7.64-7.61 (m, 1H),7.58-7.52 (m, 2H), 7.52-7.46 (m, 2H), 4.76 (s, 2H), 4.50 (s, 2H), 2.74(t, J=7.3 Hz, 2H), 1.69-1.54 (m, 2H), 1.46-1.37 (m, 2H), 0.90 (t, J=7.3Hz, 3H). ESI-MS (m/z): 428.1 [M+H]⁺

2-(((butyl(l1-oxidanyl)-l3-sulfanyl)methyl)thio)-4-(3-(hydroxymethyl)phenyl)-6-(thiazol-2-yl)nicotinonitrile.Followed standard oxidation procedure using2-(((butylthio)methyl)thio)-4-(3-(hydroxymethyl)phenyl)-6-(thiazol-2-yl)nicotinonitrileas the starting material. Isolated product in 88% yield. ¹H NMR (400MHz, CDCl₃) δ 8.08 (s, 1H), 7.96 (d, J=3.1 Hz, 1H), 7.68-7.64 (m, 1H),7.58-7.53 (m, 2H), 7.53-7.48 (m, 2H), 4.77 (s, 2H), 4.71 (d, J=13.1 Hz,1H), 4.36 (d, J=13.1 Hz, 1H), 2.96 (dt, J=13.0, 8.2 Hz, 1H), 2.81 (dt,J=13.0, 7.3 Hz, 1H), 1.82 (p, J=7.7 Hz, 2H), 1.58-1.40 (m, 2H), 0.94 (t,J=7.3 Hz, 3H). ESI-MS (m/z): 444.1 [M+H]⁺.

SW209418(3-(3-amino-2-(butyl(l1-oxidanyl)-l3-sulfanyl)-6-(thiazol-2-yl)thieno[2,3-b]pyridin-4-yl)phenyl)methanol.Followed procedure for SW209510 using2-(((butyl(l1-oxidanyl)-l3-sulfanyl)methyl)thio)-4-(3-(hydroxymethyl)phenyl)-6-(thiazol-2-yl)nicotinonitrileas the starting material to give an isolated product in 68% yield. ¹HNMR (400 MHz, CDCl₃) δ 8.01 (s, 1H), 7.88 (d, J=3.1 Hz, 1H), 7.55-7.30(m, 5H), 4.75 (s, 2H), 4.62 (s, 2H), 3.26 (ddd, J=12.8, 9.1, 6.0 Hz,1H), 3.09 (ddd, J=12.8, 9.2, 6.5 Hz, 1H), 1.76-1.61 (m, 2H), 1.51-1.38(m, 2H), 0.92 (t, J=7.3 Hz, 3H). ESI-MS (m/z): 444.1 [M+H]⁺.

Methyl3-(2-(((butyl(l1-oxidanyl)-l3-sulfanyl)methyl)thio)-3-cyano-6-(thiazol-2-yl)pyridin-4-yl)benzoate.Followed standard oxidation procedure, using methyl3-(2-(((butylthio)methyl)thio)-3-cyano-6-(thiazol-2-yl)pyridin-4-yl)benzoateas the starting material. This gave an isolated product in 86% yield. ¹HNMR (400 MHz, CDCl₃) δ 8.27 (t, J=1.6 Hz, 1H), 8.17 (dt, J=7.9, 1.4 Hz,1H), 8.09 (s, 1H), 7.97 (d, J=3.1 Hz, 1H), 7.82 (ddd, J=7.7, 1.9, 1.1Hz, 1H), 7.61 (m, 1H), 7.58 (d, J=3.1 Hz, 1H), 4.72 (d, J=13.1 Hz, 1H),4.42 (d, J=13.1 Hz, 1H), 3.92 (s, 3H), 2.95 (dt, J=13.0, 8.1 Hz, 1H),2.83 (dt, J=13.0, 7.3 Hz, 1H), 1.81 (p, J=7.7 Hz, 2H), 1.57-1.36 (m,2H), 0.92 (t, J=7.3 Hz, 3H). ESI-MS (m/z): 472.1 [M+H]⁺.

SW209416. Methyl3-(3-amino-2-(butyl(l1-oxidanyl)-l3-sulfanyl)-6-(thiazol-2-yl)thieno[2,3-b]pyridin-4-yl)benzoate.Followed procedure for SW209510 using methyl3-(2-(((butyl(l1-oxidanyl)-l3-sulfanyl)methyl)thio)-3-cyano-6-(thiazol-2-yl)pyridin-4-yl)benzoateas the starting material to give an isolated product in 68% yield. ¹HNMR (400 MHz, CDCl₃) δ 8.26-8.11 (m, 2H), 8.02 (s, 1H), 7.89 (d, J=3.2Hz, 1H), 7.76-7.56 (m, 2H), 7.49 (d, J=3.1 Hz, 1H), 4.54 (s, 2H), 3.93(s, 3H), 3.27 (ddd, J=12.8, 9.0, 6.2 Hz, 1H), 3.09 (ddd, J=12.8, 9.0,6.7 Hz, 1H), 1.79-1.61 (m, 2H), 1.55-1.39 (m, 2H), 0.92 (t, J=7.3 Hz,3H). ESI-MS (m/z): 472.1 [M+H]⁺.

Standard Hydrolysis Procedure of Ester to Carboxylic Acid:3-(2-(((butylthio)methyl)thio)-3-cyano-6-(thiazol-2-yl)pyridin-4-yl)benzoicacid. THF (214.3 μL), MeOH (214.3 μL), and H₂O (71.4 μL) were added tomethyl3-(2-(((butylthio)methyl)thio)-3-cyano-6-(thiazol-2-yl)pyridin-4-yl)benzoate(50 mg, 0.110 mmol), and last LiOH (7.9 mg, 0.329 mmol) was added. Thesolution was stirred at room temperature for 3 hours. The reactionmixture was diluted with EtOAc and washed with 1M HCl. The organic layerwas dried over Na₂SO₄, filtered, and concentrated under reducedpressure. The resulting product gave a 94% yield. ¹H NMR (400 MHz,CDCl₃) δ 8.41 (t, J=1.7 Hz, 1H), 8.26 (dt, J=8.0, 1.3 Hz, 1H), 8.13 (s,1H), 8.02 (d, J=3.1 Hz, 1H), 7.94-7.89 (m, 1H), 7.65 (t, J=7.8 Hz, 1H),7.59 (d, J=3.1 Hz, 1H), 4.53 (s, 2H), 2.75 (t, J=7.3 Hz, 2H), 1.64 (p,J=7.5 Hz, 2H), 1.43 (m, 2H), 0.91 (t, J=7.3 Hz, 3H). ESI-MS (m/z): 442.1[M+Z]⁺.

Standard amide bond coupling procedure:3-(2-(((butylt^(hi)o)methyl)thio)-3-cyano-6-(thiazol-2-yl)pyridin-4-yl)-N,N-dimethylbenzamide.Dimethylamine hydrochloride (9.25 mg, 0.114 mmol) was added to asolution of3-(2-(((butylthio)methyl)thio)-3-cyano-6-(thiazol-2-yl)pyridin-4-yl)benzoicacid (45.6 mg, 0.103 mmol), HATU (43.2 mg, 0.114 mmol), and DMF (266 μL)followed by DIPEA (36 μL, 0.21 mmol). The solution was stirred at roomtemperature for 3 hours, then diluted with EtOAc and washed with water.The organic layer was dried over Na₂SO₄, filtered, and concentratedunder reduced pressure. The isolated solid gave an 86% yield. ¹H NMR(400 MHz, CDCl₃) δ 7.99 (s, 1H), 7.97 (d, J=3.1 Hz, 1H), 7.69-7.61 (m,2H), 7.58-7.51 (m, 3H), 4.50 (s, 2H), 3.11 (s, 3H), 3.03 (s, 3H), 2.73(t, J=7.3 Hz, 2H), 1.62 (p, J=7.4 Hz, 2H), 1.40 (h, J=7.3 Hz, 2H), 0.89(t, J=7.3 Hz, 3H). ESI-MS (m/z): 469.1 [M+H]⁺.

3-(2-(((butyl(l1-oxidanyl)-l3-sulfanyl)methyl)thio)-3-cyano-6-(thiazol-2-yl)pyridin-4-yl)-N,N-dimethylbenzamide.Followed standard oxidation procedure, using3-(2-(((butylthio)methyl)thio)-3-cyano-6-(thiazol-2-yl)pyridin-4-yl)-N,N-dimethylbenzamideas the starting material to give the isolated product in 96% yield. ¹HNMR (400 MHz, Chloroform-d) δ 8.08 (s, 1H), 7.98 (d, J=3.1 Hz, 1H),7.70-7.64 (m, 2H), 7.61-7.54 (m, 3H), 4.70 (d, J=13.1 Hz, 1H), 4.42 (d,J=13.1 Hz, 1H), 3.11 (s, 3H), 3.03 (s, 3H), 2.95 (dt, J=12.9, 8.2 Hz,1H), 2.81 (dt, J=12.9, 7.2 Hz, 1H), 1.82 (p, J=7.7 Hz, 2H), 1.56-1.36(m, 2H), 0.93 (t, J=7.3 Hz, 3H). ESI-MS (m/z): 485.1 [M+H]⁺.

SW209417.3-(3-amino-2-(butyl(l1-oxidanyl)-l3-sulfanyl)-6-(thiazol-2-yl)thieno[2,3-b]pyridin-4-yl)-N,N-dimethylbenzamide.Followed procedure for SW209510 using3-(2-(((butyl(l1-oxidanyl)-l3-sulfanyl)methyl)thio)-3-cyano-6-(thiazol-2-yl)pyridin-4-yl)-N,N-dimethylbenzamideas the starting material to give the isolated product in 63% yield. ¹HNMR (400 MHz, Chloroform-d) δ 8.02 (s, 1H), 7.90 (d, J=3.1 Hz, 1H),7.62-7.51 (m, 4H), 7.49 (d, J=3.1 Hz, 1H), 4.59 (s, 2H), 3.27 (ddd,J=12.8, 9.0, 6.1 Hz, 1H), 3.15-2.97 (m, 7H), 1.78-1.64 (m, 2H),1.55-1.39 (m, 2H), 0.93 (t, J=7.3 Hz, 3H).

SW209419.3-(3-amino-2-(butyl(l1-oxidanyl)-l3-sulfanyl)-6-(thiazol-2-yl)thieno[2,3-b]pyridin-4-yl)benzoicacid. Using SW209416 as the starting material, follow the standardhydrolysis procedure of ester to carboxylic acid. This gave an isolatedyield of 98%. ¹H NMR (400 MHz, (CD₃)₂CO)) δ 8.28-8.18 (m, 2H), 8.07 (s,1H), 7.98 (d, J=3.2 Hz, 1H), 7.90 (d, J=7.6 Hz, 1H), 7.82 (d, J=3.2 Hz,1H), 7.76 (t, J=7.6 Hz, 1H), 4.82 (s, 2H), 3.20 (ddd, J=12.8, 8.8, 6.3Hz, 1H), 3.09 (ddd, J=12.9, 8.8, 6.8 Hz, 1H), 1.76-1.66 (m, 2H),1.54-1.43 (m, 2H), 0.92 (t, J=7.3 Hz, 3H). ESI-MS (m/z): 458.1 [M+H]⁺.

SW209420.(3-(3-amino-2-(butyl(l1-oxidanyl)-l3-sulfanyl)-6-(thiazol-2-yl)thieno[2,3-b]pyridin-4-yl)phenyl)(4-methylpiperazin-1-yl)methanone.Followed the standard amide bond coupling procedure, using SW209419 asthe starting material and 1-methylpiperazine as the substrate. Theproduct was purified using automated flash chromatography, recovering38% isolated yield. ¹H NMR (400 MHz, CDCl₃) δ 8.04 (s, 1H), 7.91 (d,J=3.2 Hz, 1H), 7.65-7.42 (m, 5H), 4.56 (s, 2H), 3.79 (m, 2H), 3.46 (m,2H), 3.28 (ddd, J=12.9, 8.9, 6.1 Hz, 1H), 3.10 (ddd, J=12.9, 9.2, 7.0Hz, 1H), 2.48 (m, 2H), 2.35 (m, 2H), 2.31 (s, 3H), 1.77-1.58 (m, 2H),1.54-1.38 (m, 2H), 0.94 (t, J=7.3 Hz, 3H). ESI-MS (m/z): 540.2 [M+H]⁺.

SW209508.N-allyl-3-(3-amino-2-(butyl(l1-oxidanyl)-l3-sulfanyl)-6-(thiazol-2-yl)thieno[2,3-b]pyridin-4-yl)benzamide.Followed the standard amide bond coupling procedure using SW209419 asthe starting material and allylamine as the substrate. The isolatedproduct gave a 92% yield. ¹H NMR (400 MHz, CDCl₃) δ 8.05-7.91 (m, 3H),7.88 (d, J=3.2 Hz, 1H), 7.68-7.53 (m, 2H), 7.48 (d, J=3.1 Hz, 1H),6.01-5.82 (m, 1H), 5.25 (d, J=17.2 Hz, 1H), 5.16 (dd, J=10.2, 1.4 Hz,1H), 4.52 (s, 2H), 4.19-3.98 (m, 2H), 3.24 (ddd, J=12.8, 9.0, 5.9 Hz,1H), 3.16-2.98 (m, 1H), 1.78-1.56 (m, 2H), 1.57-1.38 (m, 2H), 0.92 (t,J=7.3 Hz, 3H). ESI-MS (m/z): 497.1 [M+H]⁺.

SW209509.3-(3-amino-2-(butyl(l1-oxidanyl)-l3-sulfanyl)-6-(thiazol-2-yl)thieno[2,3-b]pyridin-4-yl)-N-(2-(dimethylamino)ethyl)benzamide.Followed the standard amide bond coupling procedure, using SW209419 asthe starting material and N,n-dimethyl-ethane-1,2-diamine as thesubstrate. The reaction mixture was diluted with EtOAc and washed withwater and NaOH was added to neutralize the pH. The organic layer wasthen dried over Na₂SO₄, filtered, and concentrated under reducedpressure. The crude product was purified using automated flashchromatography (93% DCM, 2% Et₃N, 5% MeOH) to give product in 70%isolated yield. ¹H NMR (400 MHz, CDCl₃) δ 8.02 (s, 1H), 8.00-7.95 (m,2H), 7.88 (d, J=3.2 Hz, 1H), 7.63-7.54 (m, 2H), 7.48 (d, J=3.2 Hz, 1H),4.55 (s, 2H), 3.59-3.50 (m, 2H), 3.25 (ddd, J=12.8, 9.0, 6.0 Hz, 1H),3.08 (ddd, J=12.8, 9.1, 6.6 Hz, 1H), 2.58 (t, J=5.9 Hz, 2H), 2.28 (s,6H), 1.77-1.61 (m, 2H), 1.53-1.43 (m, 2H), 0.92 (t, J=7.3 Hz, 3H).ESI-MS (m/z): 528.2 [M+H]⁺.

2,6-dichloropyridin-3-amine. The acetone/water mixture (297 mL, 5:1) wasadded to 2,6-dichloro-3-nitropyridine (3.0 g, 0.016 mol) followed by Zn(10.17 g, 0.1550 mol) and NH₄Cl (12.44 g, 0.2325 mol). The solutionstirred at room temperature overnight. The reaction mixture was thenfiltered through celite and the filtrate was extracted with EtOAc. Withthe help of brine, the organic layer was separated, dried over MgSO₄ andconcentrated under reduced pressure. ¹H NMR (400 MHz, CDCl₃) δ 7.07 (d,J=8.2 Hz, 1H), 7.02 (d, J=8.3 Hz, 1H), 4.11 (s, 2H). ESI-MS (m/z):163.0.

Potassium Ethyl Xanthate. A potassium ethoxide solution was prepared bydissolving KOH (6.5 g, 0.12 mol) in EtOH (63.4 mL). Carbon disulfide(7.14 mL, 0.118 mol) was added to the solution slowly with continuousstirring. The reaction mixture was cooled down to 5° C., filtered, andthe precipitate was recrystallized twice from warm ethanol.

5-Chlorothiazolo[5,4-b]pyridine-2-thiol. Potassium ethyl xanthate (1.9g, 0.012 mol) and anhydrous N-methyl-2-pyrrolidone (14.1 mL) were addedto 2,6-dichloropyridin-3-amine (1.0 g, 0.0061 mol) under N₂. Thesolution was refluxed (170° C.) for 3.5 hours. The reaction mixture wascooled down to room temperature, acidified to pH 5 using AcOH, dilutedin EtOAc, and washed several times with H₂O. The organic layer wasseparated, dried over MgSO₄, and concentrated under reduced pressure.This gave a red solid in 18% yield. ¹H NMR (400 MHz, Chloroform-d) δ7.41 (d, J=8.4 Hz, 1H), 7.30 (d, J=8.4 Hz, 1H). ESI-MS (m/z): 202.9.

2-(Butylthio)-5-chlorothiazolo[5,4-b]pyridine. K₂CO₃ (75 mg, 0.54 mmol),1-bromobutane (53.3 μ_(L), _(0.)493 mmol), 18-Crown-6 (13.2 mg, 0.0493mmol), and DMF (3.4 mL) were added to5-chlorothiazolo[5,4-b]pyridine-2-thiol and the solution was heated at80° C. for 3 hours. The solution was diluted with ^(E)tOAc, washed withH₂O, and the organic layer was separated, dried over MgSO₄, andconcentrated under reduced pressure. The crude product was purifiedusing flash chromatography to give 76% isolated yield. ¹H NMR (400 MHz,CDCl₃) δ 7.93 (d, J=8.5 Hz, 1H), 7.30 (d, J=8.₅ Hz, 1H), 3.31 (t, J=7.3Hz, 2H), 1.76 (p, J=7.5 Hz, 2H), 1.52-1.39 (m, 2H), 0.93 (t, J=7.3 Hz,3H). ESI-MS (m/z): 259.0 [M+H]⁺.

2-(Butylthio)-5-(thiophen-2-yl)thiazolo[5,4-b]pyridine. 2-Thienylboronicacid (49.4 mg, 0.386 mmol), CsCO₃ (126 mg, 0.386 mmol), Pd(dppf)Cl₂(15.8 mg, 0.0193 mmol), CuCl (19.1 mg, 0.193 mmol) and DMF (1 mL) wereadded to 2-(butylthio)-5-chlorothiazolo[5,4-b]pyridine (50 mg, 0.19mmol) under N₂. The reaction mixture was heated to 100° C. for 30minutes. Then the N₂ was disconnected and the vial was capped and sealedwith teflon tape and allowed to stir overnight. The reaction mixture wasdiluted in EtOAc, washed with H₂O, and the organic layer was separated,dried over MgSO₄, filtered, and concentrated under reduced pressure togive the product in 31% isolated yield. ¹H NMR (400 MHz, CDCl₃) δ 7.98(d, J=8.6 Hz, 1H), 7.63 (dd, J=3.8, 1.1 Hz, 1H), 7.51 (dd, J=5.1, 1.1Hz, 1H), 7.23 (d, J=8.6 Hz, 1H), 7.14 (dd, J=5.0, 3.8 Hz, 1H), 3.24 (t,J=7.3 Hz, 2H), 1.78-1.66 (m, 2H), 1.57-1.41 (m, 2H), 0.96 (t, J=7.4 Hz,3H). ESI-MS (m/z): 307.0 [M+H]⁺.

SW208599.2-(butyl(l1-oxidanyl)-l3-sulfanyl)-5-(thiophen-2-yl)thiazolo[5,4-b]pyridine.CHCl₃ (142 μL), AcOH (142 μL), and H₂O₂ (12.0 μL, 0.118 mmol, 30%solution in H₂O) were added to2-(butylthio)-5-(thiophen-2-yl)thiazolo[5,4-b]pyridine (18 mg. 0.059mmol) and heated at 35° C. for 2.5 hours. The solution was diluted withEtOAc and washed with saturated NaHCO₃. The organic layer was separated,dried over MgSO₄, filtered, and concentrated under reduced pressure togive product in 60% isolated yield. ¹H NMR (400 MHz, CDCl₃) δ 8.39 (d,J=8.4 Hz, 1H), 8.06 (d, J=8.4 Hz, 1H), 7.74 (dd, J=3.8, 1.1 Hz, 1H),7.61 (dd, J=5.0, 1.1 Hz, 1H), 7.19 (dd, J=5.0, 3.8 Hz, 1H), 3.15 (ddd,J=13.3, 9.8, 6.0 Hz, 1H), 2.96 (ddd, J=13.3, 9.9, 4.9 Hz, 1H), 1.98-1.80(m, 1H), 1.60-1.52 (m, 1H), 1.52-1.37 (m, 2H), 0.93 (t, J=7.2 Hz, 3H).ESI-MS (m/z): 323.0 [M+H]⁺.

2-(((Isopropylthio)methyl)thio)-6-(oxazol-2-yl)-4-phenylnicotinonitrile.Follow the standard alkylation procedure, using(chloromethyl)(isopropyl)sulfane as the alkylating substrate, and6-(oxazol-2-yl)-4-phenyl-2-thioxo-1,2-dihydropyridine-3-carbonitrile asthe starting material. The crude product was purified using flashchromatography to give a solid in 62% isolated yield. ¹H NMR (400 MHz,CDCl₃) δ 7.97 (s, 1H), 7.88 (d, J=0.7 Hz, 1H), 7.67-7.61 (m, 2H),7.56-7.50 (m, 3H), 7.37 (d, J=0.8 Hz, 1H), 4.63 (s, 2H), 3.24 (hept,J=6.7 Hz, 1H), 1.35 (d, J=6.7 Hz, 6H). ESI-MS (m/z): 368.0 [M+H]⁺.

2-(((Isopropyl(l1-oxidanyl)-l3-sulfanyl)methyl)thio)-6-(oxazol-2-yl)-4-phenylnicotinonitrile.Follow the standard oxidation procedure using2-(((isopropylthio)methyl)thio)-6-(oxazol-2-yl)-4-phenylnicotinonitrileas the starting material. Recovered quantitative isolated yield. ¹H NMR(400 MHz, CDCl₃) δ 8.03 (s, 1H), 7.88 (d, J=0.7 Hz, 1H), 7.67-7.61 (m,2H), 7.58-7.50 (m, 3H), 7.39 (d, J=0.7 Hz, 1H), 4.79 (d, J=13.3 Hz, 1H),4.55 (d, J=13.3 Hz, 1H), 3.18 (hept, J=6.9 Hz, 1H), 1.42 (d, J=1.5 Hz,3H), 1.40 (d, J=1.3 Hz, 3H). ESI-MS (m/z): 384.1 [M+H]⁺.

Standard Final Cyclization Procedure: SW208660.2-(Isopropyl(l1-oxidanyl)-l3-sulfanyl)-6-(oxazol-2-yl)-4-phenylthieno[2,3-b]pyridin-3-amine.DMF (485 μL) and MeOH (244 μL) were added to2-(((isopropyl(l1-oxidanyl)-l3-sulfanyl)methyl)thio)-6-(oxazol-2-yl)-4-phenylnicotinonitrile(47.2 mg, 0.123 mmol) dissolving it completely before KOH (4.1 mg in 100μL of H₂O) was added to the solution. The reaction mixture was stirredat 35° C. for 40 minutes. The reaction mixture was diluted with EtOAc,washed with 10% AcOH and then washed with H₂O multiple times. Theorganic layer was separated, dried over MgSO₄, filtered, andconcentrated under reduced pressure. The crude product was purifiedusing flash chromatography to give product in 40% isolated yield ¹H NMR(400 MHz, CDCl₃) δ 7.99 (s, 1H), 7.85 (d, J=0.7 Hz, 1H), 7.56-7.44 (m,5H), 7.34 (d, J=0.8 Hz, 1H), 4.69 (s, 2H), 3.41 (hept, J=6.8 Hz, 1H),1.44 (d, J=6.8 Hz, 3H), 1.28 (d, J=6.9 Hz, 3H). ESI-MS (m/z): 384.1[M+H]⁺.

2-Chloro-4-methyl-6-morpholinonicotinonitrile. Anhydrous MeOH (3.97 mL)was added to 2,6-dichloro-4-methylnicotinonitrile (500 mg, 2.67 mmol)under N₂ and the mixture was cooled down to 0° C. Morpholine (473.7 μL,5.493 mmol) was added dropwise to the solution and the solution stirredat room temperature overnight. The reaction mixture was filtered,washing the precipitate with MeOH (500 μL) and H₂O (3-4 mL). DCM wasadded to the precipitate, followed by MgSO₄, and the solution wasfiltered, then concentrated under reduced pressure. The crude productwas purified using automated flash chromatography to give product in 85%isolated yield. ¹H NMR (400 MHz, CDCl₃) δ 7.26 (s, 1H), 3.81-3.73 (m,4H), 3.68-3.58 (m, 4H), 2.42 (d, J=0.8 Hz, 3H). ESI-MS (m/z): 238.1[M+H]⁺.

4-Methyl-6-morpholino-2-thioxo-1,2-dihydropyridine-3-carbonitrile. NaOME(73.6 mg, 1.36 mmol) and methyl 3-mercaptopropionate (151 μL, 1.363mmol) were added to a solution of2-chloro-4-methyl-6morpholinonicotinonitrile (324 mg, 1.36 mmol) in DMF(4.10 mL) and the reaction mixture was stirred at 80° C. for 1 hour.Once cooled down, the reaction mixture was diluted with EtOAc and washedwith H₂O. The organic layer was separated, dried over MgSO4, filtered,and concentrated under reduced pressure to give a crude mixture of 1:1starting material to product, which was carried forward to the nextstep. ESI (m/z): 322.1 [M+H]+. NaH (150.8 mg, 3.769 mmol, 60% in mineraloil) and THF (10 mL) were added to a flame dried flask under N2,followed by the crude product from the previous step dissolved in THF(10 mL). The reaction mixture was refluxed for 6 hours and addition NaH(2 eq) was and left refluxing overnight. EtOH (1.5 mL) was added thenthe reaction mixture was concentrated down under reduced pressure. H₂O(8 mL) was added and the solution was adjusted to pH 6 with concentratedHCl before filtering to leave a crude solid that was carried forward.ESI (m/z): 236.1 [M+H]+.

4-Methyl-6-morpholino-2-(((propylthio)methyl)thio)nicotinonitrile.Followed the standard alkylating procedure, using4-methyl-6-morpholino-2-thioxo-1,2-dihydropyridine-3-carbonitrile as thestarting material and (chloromethyl)(propyl)sulfane as the alkylatingsubstrate. The crude product was carried forward. ESI (m/z): 324.1[M+H]+.

2-((((l1-oxidanyl)(propyl)-l3-sulfanyl)methyl)thio)-4-methyl-6-morpholinonicotinonitrile.Followed the standard oxidation procedure using4-methyl-6-morpholino-2-(((propylthio)methyl)thio)nicotinonitrile as thestarting material. The crude product was carried forward. ESI (m/z):340.1 [M+H]⁺.

SW208663.2-((l1-oxidanyl)(propyl)-l3-sulfanyl)-4-methyl-6-morpholinothieno[2,3-b]pyridin-3-amine.Followed the standard final cyclization procedure using2-((((l1-oxidanyl)(propyl)-l3-sulfanyl)methyl)thio)-4-methyl-6-morpholinonicotinonitrileas the starting material. The crude product was purified by flashchromatography, and PTLC to give isolated product in 10% yield. ¹H NMR(400 MHz, CDCl₃) δ 6.36 (s, 1H), 4.91 (s, 2H), 3.85-3.76 (m, 4H),3.63-3.58 (m, 4H), 3.32-3.18 (m, 1H), 3.09-2.99 (m, 1H), 2.65 (s, 3H),1.81-1.66 (m, 2H), 1.07 (t, J=7.4 Hz, 3H). ESI (m/z): 340.1 [M+H]⁺.

4-Methyl-6-(thiazol-2-yl)-2-thioxo-1,2-dihydropyridine-3-carbonitrile.Followed same procedure as4-Methyl-6-morpholino-2-thioxo-1,2-dihydropyridine-3-carbonitrile, usingmethyl 3-((3-cyano-4-methyl-6-(thiazol-2-yl)pyridin-2-yl)thio)propanoateas the starting material. The crude product was carried forward. ESI-MS(m/z): 234.0 [M+H]⁺.

4-Methyl-2-(((propylthio)methyl)thio)-6-(thiazol-2-yl)nicotinonitrile.Followed the standard alkylation procedure using4-Methyl-6-(thiazol-2-yl)-2-thioxo-1,2-dihydropyridine-3-carbonitrile asthe starting material, and (chloromethyl)(propyl)sulfane as thealkylating substrate. The crude product was purified using automatedflash chromatography to give product in 23% isolated yield. ¹H NMR (400MHz, Chloroform-d) δ 7.96 (d, J=3.1 Hz, 1H), 7.83 (s, 1H), 7.54 (d,J=3.1 Hz, 1H), 4.47 (s, 2H), 2.70 (t, J=7.2 Hz, 2H), 2.55 (s, 3H), 1.67(h, J=7.3 Hz, 2H), 0.99 (t, J=7.3 Hz, 3H). ESI-MS (m/z): 322.0 {M+H]⁺.

2-((((l1-oxidanyl)(propyl)-l3-sulfanyl)methyl)thio)-4-methyl-6-(thiazol-2-yl)nicotinonitrile.Followed the standard oxidation procedure using4-methyl-2-(((propylthio)methyl)thio)-6-(thiazol-2-yl)nicotinonitrile asthe starting material to give white solid in 91% isolated yield. ¹H NMR(400 MHz, CDCl₃) δ 7.97 (d, J=3.1 Hz, 1H), 7.92 (s, 1H), 7.56 (d, J=3.2Hz, 1H), 4.74 (d, J=13.2 Hz, 1H), 4.44 (d, J=13.1 Hz, 1H), 2.89 (m, 2H),2.57 (s, 3H), 1.93-1.79 (m, 2H), 1.06 (t, J=7.4 Hz, 3H). ESI-MS (m/z):338.0 [M+H]⁺.

SW208661.2-((l1-oxidanyl)(propyl)-l3-sulfanyl)-4-methyl-6-(thiazol-2-yl)thieno[2,3-b]pyridin-3-amine.Followed standard final cyclization procedure using2-((((l1-oxidanyl)(propyl)-l3-sulfanyl)methyl)thio)-4-methyl-6-(thiazol-2-yl)nicotinonitrileas the starting material. Purified the crude product using flashchromatography to give 61% bright green isolated product. ¹H NMR (400MHz, CDCl₃) δ 7.95 (s, 1H), 7.93 (d, J=3.2 Hz, 1H), 7.48 (d, J=3.2 Hz,1H), 5.16 (s, 2H), 3.37-3.23 (m, 1H), 3.16-3.05 (m, 1H), 2.85 (s, 3H),1.83 (h, J=7.5 Hz, 2H), 1.11 (t, J=7.4 Hz, 3H). ESI-MS (m/z): 338.0[M+H]⁺.

2-(((isopropylthio)methyl)thio)-4-methyl-6-(thiazol-2-yl)nicotinonitrile.Followed standard alkylating procedure using(chloromethyl)(isopropyl)sulfane as the alkylating substrate and4-methyl-6-(thiazol-2-yl)-2-thioxo-1,2-dihydropyridine-3-carbonitrile asthe starting material. Purified using automated flash chromatography togive product in 32% isolated yield. ¹H NMR (400 MHz, CDCl3) δ 7.94 (d,J=3.2 Hz, 1H), 7.82 (s, 1H), 7.52 (d, J=3.2 Hz, 1H), 4.48 (s, 2H), 3.18(hept, J=6.6 Hz, 1H), 2.54 (s, 3H), 1.31 (d, J=6.7 Hz, 6H). ESI-MS(m/z): 322.0 [M+H]+.

2-(((isopropyl(l1-oxidanyl)-l3-sulfanyl)methyl)thio)-4-methyl-6-(thiazol-2-yl)nicotinonitrile.Followed standard oxidation procedure using2-(((isopropylthio)methyl)thio)-4-methyl-6-(thiazol-2-yl)nicotinonitrileas the starting material to give a solid product in 84% yield. ¹H NMR(400 MHz, CDCl₃) δ 7.97 (d, J=3.1 Hz, 1H), 7.91 (s, 1H), 7.56 (d, J=3.1Hz, 1H), 4.57 (d, J=13.3 Hz, 1H), 4.46 (d, J=13.3 Hz, 1H), 3.05 (hept,J=6.9 Hz, 1H), 2.57 (s, 3H), 1.38 (d, J=7.1 Hz, 3H), 1.36 (d, J=6.7 Hz,3H). ESI-MS (m/z): 338.0 [M+H]⁺.

SW208664.2-(Isopropyl(l1-oxidanyl)-l3-sulfanyl)-4-methyl-6-(thiazol-2-yl)thieno[2,3-b]pyridin-3-amine.Followed standard final cyclization procedure using2-(((isopropyl(l1-oxidanyl)-l3-sulfanyl)methyl)thio)-4-methyl-6-(thiazol-2-yl)nicotinonitrileas the starting material. The crude product was purified using flashchromatography to give bright green oil/solid in 48% isolated yield. ¹HNMR (400 MHz, Chloroform-d) δ 7.93 (s, 1H), 7.92 (d, J=3.2 Hz, 1H), 7.46(d, J=3.2 Hz, 1H), 3.38 (hept, J=6.9 Hz, 1H), 2.84 (s, 3H), 1.46 (d,J=6.8 Hz, 3H), 1.29 (d, J=6.8 Hz, 3H). ESI-MS (m/z): 338.0 [M+H]⁺.

Ethyl 2,4-dioxo-4-(thiophen-2-yl)butanoate. 2-Acetylthiophene (1.71 mL,0.0159 mol) was added to a solution of NaOEt (730 mg Na cubes in 50 mLof EtOH) and the solution was cooled to 0° C. for 1-2 hours then diethyloxylate (3.2 ^(m)L) was added to the solution. This was left to stir atroom temperature overnight. The reaction mixture was diluted with EtOAcand H₂O with a little brine to assist the separation. The organic layerwas collected, dried with MgSO₄, filtered, and concentrated underreduced pressure. The crude product was purified using automated flashchromatography giving an oil product with 23% yield. ESI-MS (m/z): 227.0[M+H]⁺.

Ethyl3-cyano-2-(((propylthio)methyl)thio)-6-(thiophen-2-yl)isonicotinate.2-Cyanothioacetamide (250.6 mg, 2.503 mmol) and ethyl2,4-dioxo-4-(thiophen-2-yl)butanoate (565.7 mg, 2.503 mmol) weredissolved in EtOH (7.46 mL) under gentle heating (40° C.), then Et₃N(174.5 μL, 1.251 mmol) was added drop wise to the stirring solution. Thereaction mixture was heated at 60° C. and after 3 hours was concentrateddown under reduced pressure and the crude product was carried forward tothe next step. Followed standard alkylating procedure using ethyl3-cyano-6-(thiophen-2-yl)-2-thioxo-1,2-dihydropyridine-4-carboxylate asthe starting material and (chloromethyl)(propyl)sulfane as thealkylating reagent. The crude product was purified twice using automatedflash chromatography (20% EtOAc, 80% hexanes) to give 34% isolatedproduct. ¹H NMR (400 MHz, CDCl₃) δ 7.70 (s, 1H), 7.60 (dd, J=3.8, 1.1Hz, 1H), 7.44 (dd, J=5.1, 1.1 Hz, 1H), 7.04 (dd, J=5.0, 3.8 Hz, 1H),4.38 (q, J=7.2 Hz, 2H), 4.32 (s, 2H), 2.59 (t, J=7.2 Hz, 2H), 1.57 (h,J=7.4 Hz, 2H), 1.36 (t, J=7.1 Hz, 3H), 0.89 (t, J=7.3 Hz, 3H). ESI-MS(m/z): 378.9 [M+H]⁺.

Ethyl2-((((l1-oxidanyl)(propyl)-l3-sulfanyl)methyl)thio)-3-cyano-6-(thiophen-2-yl)isonicotinate.Followed standard oxidation procedure using ethyl3-cyano-2-(((propylthio)methyl)thio)-6-(thiophen-2-yl)isonicotinate asthe starting material to give a solid with quantitative yield. ¹H NMR(400 MHz, CDCl₃) δ 7.86 (s, 1H), 7.72 (dd, J=3.8, 1.1 Hz, 1H), 7.53 (dd,J=5.0, 1.1 Hz, 1H), 7.11 (dd, J=5.0, 3.8 Hz, 1H), 4.68 (d, J=13.2 Hz,1H), 4.49 (d, J=13.2 Hz, 1H), 4.43 (q, J=7.1 Hz, 2H), 2.96-2.82 (m, 2H),1.87-1.75 (m, 2H), 1.40 (t, J=7.2 Hz, 3H), 1.02 (t, J=7.4 Hz, 3H).ESI-MS (m/z): 394.9 [M+H]⁺.

SW208781. Ethyl2-((l1-oxidanyl)(propyl)-l3-sulfanyl)-3-amino-6-(thiophen-2-yl)thieno[2,3-b]pyridine-4-carboxylate.t-BuOK (74.1 mg, 0.661 mmol) was added to a solution of ethyl2-((((l1-oxidanyl)(propyl)-l3-sulfanyl)methyl)thio)-3-cyano-6-(thiophen-2-yl)isonicotinate(433.7 mg, 1.101 mmol) in DMF (4.3 mL) and the solution stirred for 40minutes at 35° C. More t-BuOK (74.1 mg, 0.661 mmol) was added andallowed to stir at 35° C. for an hour. The reaction mixture was dilutedwith EtOAc and washed with 10% AcOH, and then multiple times with H₂O.The organic layer was separated, dried over MgSO₄, filtered, andconcentrated under reduced pressure. The crude product was purifiedusing flash chromatography to give product in 30% isolated yield. ¹H NMR(400 MHz, CDCl₃) δ 8.01 (s, 1H), 7.69 (dd, J=3.8, 1.1 Hz, 1H), 7.46 (dd,J=5.0, 1.1 Hz, 1H), 7.11 (dd, J=5.0, 3.8 Hz, 1H), 6.09 (s, 2H), 4.49 (q,J=7.1 Hz, 2H), 3.36-3.22 (m, 1H), 3.15-3.00 (m, 1H), 1.87-1.68 (m, 2H),1.47 (t, J=7.1 Hz, 3H), 1.07 (t, J=7.4 Hz, 3H). ESI-MS (m/z): 394.9[M+H]⁺.

SW208782.2-((l1-oxidanyl)(propyl)-l3-sulfanyl)-3-amino-6-(thiophen-2-yl)thieno[2,3-b]pyridine-4-carboxylicacid. Followed the standard hydrolysis procedure using SW208781 as thestarting material which gave product in 40% isolated yield. ¹H NMR (400MHz, C₃D₇NO) δ 8.56 (s, 1H), 8.29 (d, J=3.7 Hz, 1H), 8.19 (s, 1H), 7.99(d, J=5.0 Hz, 1H), 7.47-7.41 (m, 1H), 3.36 (ddd, J=12.8, 8.4, 6.1 Hz,1H), 3.24 (ddd, J=12.8, 8.6, 6.8 Hz, 1H), 1.98-1.85 (m, 2H), 1.23 (t,J=7.4 Hz, 3H). ESI-MS (m/z): 366.8.

2-(((isopropylthio)methyl)thio)-4-phenyl-6-(thiazol-2-yl)nicotinonitrile.Followed the standard alkylation procedure using4-phenyl-6-(thiazol-2-yl)-2-thioxo-1,2-dihydropyridine-3-carbonitrile asthe starting material and (chloromethyl)(isopropyl)sulfane as thealkylating reagent. This was purified using automated flashchromatography to give product in 72% isolated yield. ¹H NMR (400 MHz,CDCl₃) δ 8.03 (s, 1H), 7.98 (d, J=3.1 Hz, 1H), 7.70-7.62 (m, 2H), 7.57(d, J=3.1 Hz, 1H), 7.56-7.48 (m, 3H), 4.56 (s, 2H), 3.24 (hept, J=6.7Hz, 1H), 1.36 (d, J=6.7 Hz, 6H). ESI-MS (m/z): 383.9.

2-(((isopropyl(l1-oxidanyl)-l3-sulfanyl)methyl)thio)-4-phenyl-6-(thiazol-2-yl)nicotinonitrile.Followed the standard oxidation procedure using2-(((isopropylthio)methyl)thio)-4-phenyl-6-(thiazol-2-yl)nicotinonitrileas the starting material. This gave white solid product in 91% yield. ¹HNMR (400 MHz, CDCl₃) δ 8.12 (s, 1H), 8.00 (d, J=3.1 Hz, 1H), 7.70-7.63(m, 2H), 7.60 (d, J=3.1 Hz, 1H), 7.58-7.51 (m, 3H), 4.63 (d, J=13.2 Hz,1H), 4.48 (d, J=13.2 Hz, 1H), 3.09 (hept, J=6.9 Hz, 1H), 1.42 (d, J=10.5Hz, 3H), 1.39 (d, J=10.5 Hz, 3H). ESI-MS (m/z): 399.9.

SW208780.2-(isopropyl(l1-oxidanyl)-l3-sulfanyl)-4-phenyl-6-(thiazol-2-yl)thieno[2,3-b]pyridin-3-amine.t-BuOK (2.5 mg, 0.023 mmol) was added to a solution of2-(((isopropyl(l1-oxidanyl)-l3-sulfanyl)methyl)thio)-4-phenyl-6-(thiazol-2-yl)nicotinonitrile(15 mg, 0.038 mmol) in DMF (148 μL), and stirred at 35° C. for 40minutes. The reaction mixture was diluted with EtOAc and washed with 10%AcOH, then several times with H₂O. The organic layer was dried overNaSO₄, filtered, and concentrated under reduced pressure. The crudeproduct was purified using flash chromatography to give product' in 75%yield. ¹H NMR (400 MHz, CDCl₃) δ 8.05 (s, 1H), 7.91 (d, J=3.2 Hz, 1H),7.57-7.42 (m, 6H), 4.68 (s, 2H), 3.47-3.33 (m, 1H), 1.44 (d, J=6.8 Hz,3H), 1.27 (d, J=6.8 Hz, 3H). ESI-MS (m/z): 399.9.

Methyl4-(2-(((butyl(l1-oxidanyl)-l3-sulfanyl)methyl)thio)-3-cyano-6-(thiazol-2-yl)pyridin-4-yl)benzoate.Followed standard oxidation procedure using methyl4-(2-(((butylthio)methyl)thio)-3-cyano-6-(thiazol-2-yl)pyridin-4-yl)benzoateas the starting material to give white solid in 98% isolated yield. ¹HNMR (400 MHz, CDCl₃) δ 8.15 (d, J=8.3 Hz, 2H), 8.05 (s, 1H), 7.95 (d,J=3.1 Hz, 1H), 7.68 (d, J=8.3 Hz, 2H), 7.57 (d, J=3.1 Hz, 1H), 4.68 (d,J=13.1 Hz, 1H), 4.42 (d, J=13.1 Hz, 1H), 3.91 (s, 3H), 3.01-2.86 (m,1H), 2.87-2.74 (m, 1H), 1.88-1.72 (m, 2H), 1.55-1.35 (m, 2H), 0.91 (t,J=7.3 Hz, 3H). ESI-MS (m/z): 472.1 [M+H]⁺.

SW209127. Methyl4-(3-amino-2-(butyl(l1-oxidanyl)-l3-sulfanyl)-6-(thiazol-2-yl)thieno[2,3-b]pyridin-4-yl)benzoate.t-BuOK (21.8 mg, 0.194 mmol) was added to methyl4-(2-(((butyl(l1-oxidanyl)-l3-sulfanyl)methyl)thio)-3-cyano-6-(thiazol-2-yl)pyridin-4-yl)benzoate(152.8 mg, 0.3239 mmol) in DMF (1.30 mL) and the solution stirred at 35°C. for 40 minutes. The reaction mixture was diluted with EtOAc andwashed with 10% AcOH, and several times with H₂O. The o^(r)ganic layerwas separated, dried over Na₂SO₄, filtered, and concentrated underreduced pressure. The crude produc_(t) was purified using automatedflash chromatograph_(y) t_(o) give the bright green product in 66%isolated yield. ¹H NMR (400 MHz, CDCl₃) δ 8.19 (d, J=7.5 Hz, 2H), 8.04(s, 1H), 7.91 (d, J=3.2 Hz, 1H), 7.67-7.54 (m, 2H), 7.50 (d, J=3.2 Hz,1H), 3.97 (s, 3H), 3.27 (ddd, J=12.8, 8.9, 6.2 Hz, 1H), 3.10 (ddd,J=12.8, 9.0, 6.8 Hz, 1H), 1.81-1.63 (m, 2H), 1.54-1.39 (m, 2H), 0.93 (t,J=7.3 Hz, 3H). ESI-MS (m/z): 472.1 [M+H]⁺.

SW209281.4-(3-amino-2-(butyl(l1-oxidanyl)-l3-sulfanyl)-6-(thiazol-2-yl)thieno[2,3-b]pyridin-4-yl)benzoicacid. Followed standard hydrolysis procedure using SW209127 as thestarting material to give bright green solid in 84% isolated yield. ¹HNMR (400 MHz, CDCl₃) δ 8.16 (d, J=8.4 Hz, 2H), 8.05 (s, 1H), 7.95 (d,J=3.2 Hz, 1H), 7.68-7.55 (m, 2H), 7.52 (d, J=3.2 Hz, 1H), 3.40-3.24 (m,1H), 3.24-3.04 (m, 1H), 1.83-1.65 (m, 2H), 1.55-1.37 (m, 2H), 0.93 (t,J=7.3 Hz, 3H). ESI-MS (m/z): 458.1 [M+H]⁺.

SW209282.4-(3-amino-2-(butyl(l1-oxidanyl)-l3-sulfanyl)-6-(thiazol-2-yl)thieno[2,3-b]pyridin-4-yl)-N,N-dimethylbenzamide.Followed standard amide bond coupling procedure using SW209281 as thestarting material and dimethylamine hydrochloride as the couplingreagent. The product was purified using automated flash chromatography(20% hexane, 80% EtOAc) to give bright green solid in 59% isolated yield¹H NMR (400 MHz, CDCl₃) δ 8.03 (s, 1H), 7.91 (d, J=3.2 Hz, 1H),7.66-7.44 (m, 5H), 3.36-3.21 (m, 1H), 3.14 (s, 3H), 3.13-3.06 (m, 1H),3.02 (s, 3H), 1.81-1.64 (m, 2H), 1.55-1.41 (m, 2H), 0.93 (t, J=7.3 Hz,3H). ESI-MS (m/z): 485.1 [M+H]⁺.

2-(((butylthio)methyl)thio)-3-cyano-6-(thiophen-2-yl)isonicotinic acid.Followed standard hydrolysis procedure using ethyl2-(((butylthio)methyl)thio)-3-cyano-6-(thiophen-2-yl)isonicotinate asthe starting material to give isolated product in 94% yield. ¹H NMR (400MHz, CDCl₃) δ 10.65 (s, 1H), 7.95 (s, 1H), 7.76 (dd, J=3.8, 1.1 Hz, 1H),7.57 (dd, J=5.0, 1.0 Hz, 1H), 7.17 (dd, J=5.0, 3.7 Hz, 1H), 4.46 (s,2H), 2.72 (t, J=7.2 Hz, 2H), 1.67-1.55 (m, 2H), 1.40 (h, J=7.4 Hz, 2H),0.90 (t, J=7.3 Hz, 3H). ESI-MS (m/z): 365.0 [M+H]⁺.

2-(((butylthio)methyl)thio)-3-cyano-N,N-dimethyl-6-(thiophen-2-yl)isonicotinamide.Followed standard amide bond coupling procedure using2-(((butylthio)methyl)thio)-3-cyano-6-(thiophen-2-yl)isonicotinic acidas the starting material and dimethylamine as the coupling reagent. Thecrude material was purified using automated flash chromatography (20%EtOAc, 80% hexanes) to give product in 53% isolated yield. ¹H NMR (400MHz, Chloroform-d) δ 7.67 (d, J=3.8 Hz, 1H), 7.54 (d, J=5.0 Hz, 1H),7.34 (s, 1H), 7.15 (t, J=4.8, 3.9, 0.7 Hz, 1H), 4.49 (s, 2H), 3.16 (s,3H), 2.98 (s, 3H), 2.72 (t, 2H), 1.62 (p, J=7.7 Hz, 2H), 1.41 (h, J=7.3Hz, 2H), 0.90 (t, J=7.7, 7.0 Hz, 3H). ESI-MS (m/z): 392.1 [M+H]⁺.

2-(((butyl(l1-oxidanyl)-l3-sulfanyl)methyl)thio)-3-cyano-N,N-dimethyl-6-(thiophen-2-yl)isonicotinamide.Followed standard oxidation procedure using2-(((butylthio)methyl)thio)-3-cyano-N,N-dimethyl-6-(thiophen-2-yl)isonicotinamideas the starting material to give solid product in 85% isolated yield. ¹HNMR (400 MHz, CDCl₃) δ 7.67 (d, J=3.8 Hz, 1H), 7.54 (d, J=5.0 Hz, 1H),7.34 (s, 1H), 7.20-7.06 (m, 1H), 4.49 (s, 2H), 3.16 (s, 3H), 2.98 (s,3H), 2.72 (t, 2H), 1.62 (p, J=7.7 Hz, 2H), 1.41 (h, J=7.3 Hz, 2H), 0.90(t, J=7.7, 7.0 Hz, 3H). ESI-MS (m/z): 408.1 [M+H]⁺.

SW209283.3-amino-2-(butyl(l1-oxidanyl)-l3-sulfanyl)-N,N-dimethyl-6-(thiophen-2-yl)thieno[2,3-b]pyridine-4-carboxamide.t-BuOK (6.5 mg, 0.058 mmol) was added to a solution of2-(((butyl(l1-oxidanyl)-l3-sulfanyl)methyl)thio)-3-cyano-N,N-dimethyl-6-(thiophen-2-yl)isonicotinamide(39.2 mg, 0.962 mmol) in DMF (380 μL) and the solution was heated at 35°C. for 40 minutes. The reaction mixture was diluted with EtOAc andwashed with 10% AcOH, then several times with H₂O. The organic layer wasseparated and dried over Na₂SO₄, filtered, and concentrated underreduced pressure. The crude material was isolated using automated flashchromatography (20% hexanes, 80% EtOAc) to give the final product in 20%isolated yield. ¹H NMR (400 MHz, Chloroform-d) δ 7.66 (dd, J=3.8, 1.1Hz, 1H), 7.52-7.42 (m, 2H), 7.13 (dd, J=5.0, 3.7 Hz, 1H), 3.34-3.23 (m,1H), 3.21 (s, 3H), 3.15-3.02 (m, 1H), 2.96 (s, 3H), 1.79-1.62 (m, 2H),1.55-1.36 (m, 2H), 0.93 (t, J=7.3 Hz, 3H). ESI-MS (m/z): 408.1 [M+H]⁺.

SW212366.4-(3-amino-2-(butylsulfinyl)-6-(thiazol-2-yl)thieno[2,3-b]pyridin-4-yl)benzyldimethylglycinate. N,N-Dimethylglycine (3.5 mg, 0.034 mmol),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (6.5 mg, 0.034 mmol), andDMAP (4.1 mg, 0.0334 mmol) were added to SW209510 (10 mg, 0.023 mmol)and dissolved in DMF (270 μL). The reaction mixture stirred at roomtemperature overnight, then neutralized with 1M NaOH, washed with H₂Oand extracted with EtOAc. The organic layer was dried over Na₂SO₄,filtered and concentrated under reduced pressure. The crude product waspurified using flash chromatography (7% MeOH, 93% DCM) to give aquantitative yield of green solid product ¹H NMR (400 MHz, CDCl₃) δ 8.02(s, 1H), 7.90 (d, J=3.2 Hz, 1H), 7.56-7.43 (m, 5H), 5.25 (s, 2H), 4.61(s, 2H), 3.35-3.27 (m, 1H), 3.26 (s, 2H), 3.16-3.04 (m, 1H), 2.37 (s,6H), 1.78-1.63 (m, 2H), 1.55-1.39 (m, 2H), 0.93 (t, J=7.3 Hz, 3H).ESI-MS (m/z): 529.1 [M+H]⁺.

Methyl 2-(4-formylphenoxy)acetate. To a solution of4-hydroxybenzaldehyde (3.0 g, 25 mmol) in acetone (61.4 mL), K₂CO₃ (5.43g, 39.3 mmol) was added and the mixture was stirred vigorously.Methylbromoacetate (2.8 mL, 29 mmol) was added and the mixture wasstirred for 3.5 hrs at room temperature. The reaction mixture wasconcentrated down under reduced pressure then washed with H₂O andextracted with EtOAc. The organic layer was separated, dried overNa₂SO₄, filtered, and concentrated to give a colorless oil thatsolidified under vacuum in 82% isolated yield. ¹H NMR (400 MHz, CDCl₃) δ9.87 (s, 1H), 7.82 (d, J=8.8 Hz, 2H), 6.98 (d, J=8.7 Hz, 2H), 4.70 (s,2H), 3.79 (s, 3H). ESI-MS (m/z): 195.1 [M+H]⁺.

Methyl (E)-2-(4-(3-oxo-3-(thiazol-2-yl)prop-1-en-1-yl) phenoxy)acetate.2-acetylthiazole (534 μL, 5.15 mmol) was added to a solution of methyl2-(4-formylphenoxy)acetate (1.0 g, 5.2 mmol) in MeOH (11 mL) under N₂.NaOMe (279 mg, 5.15 mmol) was added last and the reaction mixturestirred at room temperature overnight. The reaction mixture wasfiltered, and the precipitate was washed with small amount of MeOH thendiluted with DCM and washed with H₂O. The organic layer was separated,dried over Na₂SO₄, filtered, and concentrated under reduced pressure.This gave solid product in 21% isolated yield. ¹H NMR (400 MHz,Chloroform-d) δ 8.03 (d, J=3.0 Hz, 1H), 7.95 (d, J=15.9 Hz, 1H), 7.82(d, J=16.0 Hz, 1H), 7.70-7.61 (m, 3H), 6.92 (d, J=8.8 Hz, 2H), 4.67 (s,2H), 3.80 (s, 3H). ESI-MS (m/z): 304.1 [M+H]⁺.

Methyl2-(4-(2-(((butylthio)methyl)thio)-3-cyano-6-(thiazol-2-yl)pyridin-4-yl)phenoxy)acetate.EtOH (495 μL) was added to 2-cyanothioacetamide (49.5 mg, 0.494 mmol)and methyl(E)-2-(4-(3-oxo-3-(thiazol-2-yl)prop-1-en-1-yl)phenoxy)acetate(50 mg, 0.16 mmol), followed by 1 drop of piperidine. The reactionmixture stirred at 80° C. for 4 hours then was concentrated underreduced pressure and the crude was carried forward to the next step.Followed the standard alkylation procedure, using methyl2-(4-(3-cyano-6-(thiazol-2-yl)-2-thioxo-1,2-dihydropyridin-4-yl)phenoxy)acetateas the starting material and butyl(chloromethyl)sulfane as thealkylating reagent. The crude product was purified using automated flashchromatography (20% EtOAc, 80% hexanes) to give solid product in 70%isolated yield. ¹H NMR (400 MHz, CDCl₃) δ 7.96 (s, 1H), 7.95 (d, J=3.1Hz, 1H), 7.62 (d, J=8.8 Hz, 2H), 7.54 (d, J=3.1 Hz, 1H), 7.02 (d, J=8.8Hz, 2H), 4.69 (s, 2H), 4.49 (s, 2H), 3.81 (s, 3H), 2.73 (t, J=7.3 Hz,2H), 1.68-1.56 (m, 2H), 1.46-1.34 (m, 2H), 0.89 (t, J=7.3 Hz, 3H).ESI-MS (m/z): 486.1 [M+H]⁺.

Methyl2-(4-(2-(((butyl(l1-oxidanyl)-l3-sulfanyl)methyl)thio)-3-cyano-6-(thiazol-2-yl)pyridin-4-yl)phenoxy)acetate.Followed the standard oxidation procedure using methyl2-(4-(2-(((butylthio)methyl)thio)-3-cyano-6-(thiazol-2-yl)pyridin-4-yl)phenoxy)acetateas the starting material. The crude product was purified using automatedflash chromatography (50% EtOAc, 50% hexanes). ¹H NMR (400 MHz, CDCl₃) δ7.97 (s, 1H), 7.90 (d, J=3.1 Hz, 1H), 7.57 (d, J=8.8 Hz, 2H), 7.52 (d,J=3.1 Hz, 1H), 6.98 (d, J=8.8 Hz, 2H), 4.65 (s, 2H), 4.62 (d, J=13.1 Hz,1H), 4.37 (d, J=13.1 Hz, 1H), 3.75 (s, 3H), 2.96-2.84 (m, 1H), 2.81-2.71(m, 1H), 1.76 (p, J=7.6 Hz, 2H), 1.51-1.33 (m, 2H), 0.88 (t, J=7.3 Hz,3H). ESI-MS (m/z): 502.1 [M+H]⁺.

SW212365. Methyl2-(4-(3-amino-2-(butyl(l1-oxidanyl)-l3-sulfanyl)-6-(thiazol-2-yl)thieno[2,3-b]pyridin-4-yl)phenoxy)acetate.Methyl2-(4-(2-(((butyl(l1-oxidanyl)-l3-sulfanyl)methyl)thio)-3-cyano-6-(thiazol-2-yl)pyridin-4-yl)phenoxy)acetate(80 mg, 0.16 mmol) and t-BuOK (10.7 mg, 0.0954 mmol) were combined in avial that was evacuated and backfilled with N₂ three times, then DMF(627 μL) was added and N₂ was bubbled through the solution. The reactionmixture was stirred at room temperature for about 10 minutes and thenwas diluted with EtOAc and washed with 10% AcOH. The organic layer waswashed several times with water, dried over Na₂SO₄, filtered, andconcentrated. The crude product was purified using automated flashchromatography (30% EtOAc, 70% hexanes) to give green solid with 56%isolated yield. ¹H NMR (400 MHz, CDCl₃) δ 7.97 (s, 1H), 7.89-7.86 (m,1H), 7.47 (d, J=3.1 Hz, 1H), 7.46-7.37 (m, 2H), 7.02 (d, J=8.5 Hz, 2H),4.70 (s, 2H), 4.66 (s, 2H), 3.82 (s, 3H), 3.34-3.18 (m, 1H), 3.16-3.01(m, 1H), 1.77-1.64 (m, 2H), 1.51-1.38 (m, 2H), 0.92 (t, J=7.3 Hz, 3H).ESI-MS (m/z): 502.1 [M+H]⁺

SW212364.2-(4-(3-amino-2-(butyl(l1-oxidanyl)-l3-sulfanyl)-6-(thiazol-2-yl)thieno[2,3-b]pyridin-4-yl)phenoxy)ethan-1-ol.Followed the same procedure as for2-(((butylthio)methyl)thio)-4-(4-(hydroxymethyl)phenyl)-6-(thiazol-2-yl)nicotinonitrileusing SW212365 as the starting material to give a quantitative yield ofdesired product ¹H NMR (400 MHz, CDCl₃) δ 8.01 (s, 1H), 7.90 (d, J=3.2Hz, 1H), 7.48 (d, J=3.1 Hz, 1H), 7.46-7.35 (m, 2H), 7.07-6.99 (m, 2H),4.69 (s, 2H), 4.18-4.11 (m, 2H), 4.04-3.96 (m, 2H), 3.34-3.23 (m, 1H),3.17-3.04 (m, 1H), 1.80-1.61 (m, 2H), 1.53-1.40 (m, 2H), 0.93 (t, J=7.3Hz, 3H). ESI-MS (m/z): 474.1 [M+H]⁺.

SW212363.2-(4-(3-amino-2-(butyl(l1-oxidanyl)-l3-sulfanyl)-6-(thiazol-2-yl)thieno[2,3-b]pyridin-4-yl)phenoxy)aceticacid. Followed the standard hydrolysis procedure using SW212365 as thestarting material to give a quantitative yield. ¹H NMR (400 MHz, MeOD) δ7.98 (s, 1H), 7.94 (d, J=3.2 Hz, 1H), 7.74 (d, J=3.2 Hz, 1H), 7.46 (d,J=8.4 Hz, 2H), 7.14 (d, J=8.9 Hz, 2H), 4.67 (s, 2H), 3.35-3.24 (m, 1H),3.16-3.04 (m, 1H), 1.78-1.57 (m, 2H), 1.55-1.43 (m, 2H), 0.95 (t, J=7.3Hz, 3H). ESI-MS (m/z): 488.1 [M+H]⁺.

Synthesis of Chloromethyl Thio Ethers

4-mercaptobutyl acetate. Porcine lipase (2.35 g) was added to a solutionof 4-mercapto-1-butanol (2.45 g, 23.10 mmol) in ethyl acetate (42.0 ml).The reaction was heated at 30° C. for 6 days. Despite incompleteconversion the mixture was filtered and condensed. Purification wascarried out on an automated flash chromatography system in 100% DCM togive oil in 84% yield. ¹H NMR (400 MHz, CHCl₃) δ 4.08 (t, J=6.2 Hz, 2H),2.57 (q, J=7.1 Hz, 2H), 2.05 (s, 3H), 1.85-1.59 (m, 4H), 1.36 (t, J=7.9Hz, 1H).

4-((chloromethyl)thio)butyl acetate. Hydrogen chloride gas was bubbledfor 40 minutes into 4-mercaptobutyl acetate (2.84 g, 19.2 mmol) whichhad been cooled in a dry ice/acetone bath and until the internaltemperature stabilized before paraformaldehyde (0.815 g, 27.17 mmol) wasslowly added using a solid addition funnel. The reaction was stirredcold for 3 hours during which hydrogen chloride bubbling was continuedand then ceased as the reaction was warmed gently to ambient temperatureand stirred overnight. The crude mixture was diluted with minimal DCM.The aqueous phase was removed and the organic layer was washed withbrine and dried over Na₂SO₄, filtered and condensed to give an oil in62% yield. ¹H NMR (400 MHz, CHCl₃) 4.75 (s, 2H), 4.10 (t, J=6.0 Hz, 2H),2.95-2.66 (m, 2H), 2.06 (s, 3H), 1.85-1.67 (m, 4H).

4-((((3-cyano-4-phenyl-6-(thiophen-2-yl)pyridin-2-yl)thio)methyl)thio)butylacetate. A mixture of 4-((chloromethyl)thio)butyl acetate (602.3 mg, 3.1mmol),4-phenyl-6-(thiophen-2-yl)-2-thioxo-1,2-dihydropyridine-3-carbonitrile(352.2 mg, 1.2 mmol) and triethylamine (250 ml, 1.8 mmol) inacetonitrile (1.2 ml) was refluxed for three hours. The crude mixturewas then condensed and purified on an automated flash chromatographysystem in 0-40% EtOAc/hexanes. Fractions containing the desired productwere further purified on an automated flash chromatography in 0-30%EtOAc/hexanes to give a clear oil in 41% yield. ¹H NMR (400 MHz, CHCl₃)δ 7.72 (dd, J=3.7, 1.1 Hz, 1H), 7.64-7.59 (m, 2H), 7.55 (dt, J=5.6, 2.3Hz, 4H), 7.44 (s, 1H), 7.17 (dd, J=5.0, 3.8 Hz, 1H), 4.55 (s, 2H),4.11-4.02 (m, 2H), 2.86-2.63 (m, 2H), 2.05 (s, 3H), 1.77 (t, J=3.4 Hz,4H). ESI-MS (m/z): 455.1 [M+H]⁺.

2-((((4-hydroxybutyl)thio)methyl)thio)-4-phenyl-6-(thiophen-2-yl)nicotinonitrile.K₂CO₃ (157.7 mg, 1.14 mmol) was added to a solution of4-((((3-cyano-4-phenyl-6-(thiophen-2-yl)pyridin-2-yl)thio)methyl)thio)butylacetate. (245.4 mg, 0.54 mmol) in methanol (8.0 ml) and water (2.0 ml)and the reaction was stirred for 2 hours. The mixture was dried thendiluted with EtOAc and washed twice with water and then brine. Theorganic layer was dried over Na₂SO₄, filtered and concentrated underreduce pressure to give desired product in 71% yield. ¹H NMR (400 MHz,CDCl₃) δ 7.72 (dd, J=3.7, 1.1 Hz, 1H), 7.61 (dd, J=6.6, 3.0 Hz, 2H),7.54 (dd, J=5.1, 2.2 Hz, 4H), 7.43 (s, 1H), 7.16 (dd, J=5.0, 3.8 Hz,1H), 4.55 (s, 2H), 3.67 (t, J=6.2 Hz, 2H), 2.80 (t, J=7.1 Hz, 2H),1.84-1.63 (m, 4H). ESI-MS (m/z): 413.1 [M+H]⁺.

4-((((3-cyano-4-phenyl-6-(thiophen-2-yl)pyridin-2-yl)thio)methyl)sulfinyl)butylacetate. Acetic acid (370 μl) and hydrogen peroxide (29 μl, 30% solutionin water) were added to the solution of4-((((3-cyano-4-phenyl-6-(thiophen-2-yl)pyridin-2-yl)thio)methyl)thio)butylacetate (85.2 mg, 0.19 mmol) in chloroform (370 μl). The reactionmixture was stirred at 32° C. for 90 min. Once complete, the reactionwas diluted with chloroform and washed with saturated NaHCO₃ solution,and extracted three times with chloroform. The combined organic layerswas dried over Na₂SO₄, filtered and concentrated under reduce pressureto give designed product in 94% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.77(d, J=3.8 Hz, 1H), 7.62 (dd, J=4.1, 2.3 Hz, 2H), 7.60-7.54 (m, 4H), 7.51(s, 1H), 7.19 (dd, J=5.0, 3.8 Hz, 1H), 4.78 (d, J=13.0 Hz, 1H), 4.44 (d,J=13.0 Hz, 1H), 4.11 (t, J=6.4 Hz, 2H), 3.03 (dt, J=12.9, 8.0 Hz, 1H),2.87 (dt, J=12.8, 7.3 Hz, 1H), 2.05 (s, 3H), 2.03-1.77 (m, 4H). ESI-MS(m/z): 471.1 [M+H]⁺.

SW209129.4-((3-amino-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-2-yl)sulfinyl)butylacetate. Potassium tert-butoxide (9.7 mg, 0.086 mmol) was added to asolution of4-((((3-cyano-4-phenyl-6-(thiophen-2-yl)pyridin-2-yl)thio)methyl)sulfinyl)butylacetate (58.2 mg, 0.12 mmol) in DMF (490 μl). The reaction mixture wasstirred at 35° C. for 45 minutes, then diluted with EtOAc and washedseveral times with water. The aqueous layer was also back-extracted. Thecombined organic layer was washed with brine, dried over Na₂SO₄,filtered and concentrated under reduce pressure. Purification wascarried out using automated flash chromatography in 0-90% EtOAc/hexanesto give the desired product in 44% yield. ¹H NMR (400 MHz, CDCl₃) δ7.63-7.49 (m, 5H), 7.45 (dd, J=4.9, 1.1 Hz, 2H), 7.41 (s, 1H), 7.10 (dd,J=5.0, 3.7 Hz, 1H), 4.59 (bs, 2H), 4.08 (t, J=5.8 Hz, 2H), 3.39-3.23 (m,1H), 3.10 (ddd, J=12.8, 8.4, 6.2 Hz, 1H), 2.03 (s, 3H), 1.96-1.72 (m,4H). ESI-MS (m/z): 471.1 [M+H]⁺.

SW209128.4-((3-amino-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-2-yl)sulfonyl)butylacetate. Isolated as the over oxidation product from4-((3-amino-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-2-yl)sulfinyl)butylacetate in 13.5% yield.

¹H NMR (400 MHz, CDCl₃) δ 7.71 (dd, J=3.7, 1.1 Hz, 1H), 7.61-7.55 (m,3H), 7.53-7.44 (m, 4H), 7.15 (dd, J=5.0, 3.7 Hz, 1H), 5.10 (s, 2H), 4.06(t, J=6.3 Hz, 2H), 3.32-3.18 (m, 2H), 2.02 (s, 3H), 1.98-1.87 (m, 2H),1.77 (dt, J=8.6, 6.4 Hz, 2H). ESI-MS (m/z): 487.1 [M+H]⁺.

SW209271.4-((3-amino-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-2-yl)sulfinyl)butan-1-ol.K₂CO₃ (12.5 mg, 0.09 mmol) was added to a solution of SW209129 (18.7 mg,0.04 mmol) in methanol (470 μl) and water (100 μl) and the reaction wasstirred for 2.5 hours. The mixture was dried then diluted with EtOAc andwashed twice with water and then brine. The organic layer was dried overNa₂SO₄, filtered and concentrated under reduce pressure to give desiredproduct in 80% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.62 (d, J=1.1 Hz, 1H),7.60-7.50 (m, 4H), 7.49-7.41 (m, 3H), 7.11 (dd, J=5.0, 3.7 Hz, 1H),4.68-4.44 (s, 2H), 3.67 (t, J=6.1 Hz, 2H), 3.42-3.27 (m, 1H), 3.13 (ddd,J=12.9, 8.4, 6.9 Hz, 1H), 1.93-1.65 (m, 4H). ESI-MS (m/z): 429.0 [M+H]⁺.

4-((((3-cyano-4-phenyl-6-(thiophen-2-yl)pyridin-2-yl)thio)methyl)thio)butylmethanesulfonate. A solution of triethylamine (38 μl, 0.28 mmol) inanhydrous DCM (1.0 ml) is cooled in an ice bath before the addition of2-((((4-hydroxybutyl)thio)methyl)thio)-4-phenyl-6-(thiophen-2-yl)nicotinonitrile(40.6 mg, 0.098 mmol) followed by dropwise addition of methanesulfonylchloride (17.5 μl, 0.23 mmol). After 30 minutes the crude mixture waswashed with brine and dried over Na₂SO₄, filtered and condensed to givedesired product in 98% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.74 (dd, J=3.9,1.1 Hz, 1H), 7.64-7.50 (m, 7H), 7.17 (dd, J=5.1, 3.8 Hz, 1H), 5.46 (s,2H), 4.01-3.78 (m, 2H), 2.65 (ddd, J=10.1, 5.3, 1.9 Hz, 2H), 2.52-2.37(m, 4H). ESI-MS (m/z): 491.1 [M+H]⁺.

2-((((4-chlorobutyl)thio)methyl)thio)-4-phenyl-6-(thiophen-2-yl)nicotinonitrile.Lithium chloride (32.0 mg, 0.75 mmol) was added to a solution of4-((((3-cyano-4-phenyl-6-(thiophen-2-yl)pyridin-2-yl)thio)methyl)thio)butylmethanesulfonate (21.8 mg, 0.044 mmol) in DMF (0.4 ml). The reactionwent to completion within two days. The mixture was diluted with EtOAcand washed several times with water and then brine. The organic layerwas dried over Na₂SO₄, filtered and condensed. Purification wasperformed on an automated chromatography system in 0-40% EtOAc/hexanesand gave the desired product in 76% yield. ¹H NMR (400 MHz, CDCl₃) δ7.72 (dd, J=3.7, 1.1 Hz, 1H), 7.62 (dd, J=6.5, 3.0 Hz, 2H), 7.59-7.52(m, 4H), 7.44 (s, 1H), 7.17 (dd, J=5.0, 3.8 Hz, 1H), 4.56 (s, 2H), 3.56(t, J=6.3 Hz, 2H), 2.80 (t, J=7.0 Hz, 2H), 1.95-1.79 (m, 4H). ESI-MS(m/z): 431.0 [M+H]⁺.

2-((((4-chlorobutyl)sulfinyl)methyl)thio)-4-phenyl-6-(thiophen-2-yl)nicotinonitrile.Acetic acid (70 μl) and hydrogen peroxide (5.2 μl, 30% solution inwater) were added to the solution of2-((((4-chlorobutyl)thio)methyl)thio)-4-phenyl-6-(thiophen-2-yl)nicotinonitrile(14.6 mg, 0.034 mmol) in chloroform (70 μl). The reaction mixture wasstirred at 32° C. for 40 min and then diluted with chloroform and waswashed with saturated NaHCO₃ solution and extracted three times withchloroform. The combined organic layers was dried over Na₂SO₄, filteredand concentrated under reduce pressure to give designed product. ¹H NMR(400 MHz, CDCl₃) δ 7.77 (d, J=3.8 Hz, 1H), 7.62 (dd, J=6.6, 2.9 Hz, 2H),7.57 (q, J=4.5, 3.1 Hz, 4H), 7.51 (s, 1H), 7.19 (t, J=4.4 Hz, 1H), 4.77(d, J=13.0 Hz, 1H), 4.47 (d, J=13.0 Hz, 1H), 3.58 (t, J=6.2 Hz, 2H),3.03 (dt, J=13.2, 7.7 Hz, 1H), 2.87 (dt, J=13.4, 7.0 Hz, 1H), 2.16-1.87(m, 4H). ESI-MS (m/z): 447.1 [M+H]⁺.

SW209329.2-((4-chlorobutyl)sulfinyl)-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-3-amine.A basic methanolic solution (1.0 mg, 0.018 mmol of potassium hydroxidein 12.0 μl water and 57.5 μl methanol) was transferred to a vialcontaining a solution of2-((((4-chlorobutyl)sulfinyl)methyl)thio)-4-phenyl-6-(thiophen-2-yl)nicotinonitrile(12.4 mg, 0.028 mmol) in dimethylformamide (91.5 μl). The reaction washeated at 38° C. for 30 minutes, before being cooled, diluted with EtOAcand washed several times with water, then brine. The organic layer wasdried over Na₂SO₄, filtered and condensed. The crude mixture waspurified using an automated chromatography system in 0-60%EtOAc/hexanes. Isolated yield=65%. ¹H NMR (400 MHz, CDCl₃) δ 7.64 (d,J=3.7 Hz, 1H), 7.61-7.50 (m, 4H), 7.50-7.43 (m, 3H), 7.12 (t, J=4.4 Hz,1H), 4.59 (s, 2H), 3.66-3.47 (m, 2H), 3.40-3.24 (m, 1H), 3.20-3.06 (m,1H), 1.95 (q, J=5.6 Hz, 4H). ESI-MS (m/z): 447.0 [M+H]⁺.

3-mercaptopropyl acetate. Porcine lipase (5.52 g) was added to asolution of 3-mercapto-1-propanol (5.03 g, 54.6 mmol) in ethyl acetate(70 ml). The reaction was heated at 28° C. for 12 days. Despiteincomplete conversion the mixture was filtered and condensed.Purification was carried out on an automated flash chromatography systemin 100% DCM to give oil in 66% yield. ¹H NMR (400 MHz, CHCl₃) δ 4.17 (t,J=6.2 Hz, 2H), 2.60 (q, J=7.4 Hz, 2H), 2.05 (s, 3H), 1.93 (p, J=6.6 Hz,2H), 1.39 (t, J=8.1 Hz, 2H).

3-((chloromethyl)thio)propane-1-thiol. Hydrogen chloride gas was bubbledfor 60 minutes into 3-((chloromethyl)thio)propane-1-thiol (4.80 g, 35.7mmol) which had been cooled in a dry ice/acetone bath and until theinternal temperature stabilized before paraformaldehyde (1.59 g, 53.3mmol) was slowly added using a solid addition funnel. The reaction wasstirred cold for 1.5 hours during which hydrogen chloride bubbling wascontinued and then ceased as the reaction was warmed gently to ambienttemperature and stirred overnight. The crude mixture was diluted withminimal DCM. The aqueous phase was removed and the organic layer waswashed with brine and dried over Na₂SO₄, filtered and condensed to givean oil in 80% yield of a mixture of 2.4:1 desired monomer chloride todiacetate dimer. ¹H NMR (400 MHz, CHCl₃) δ 4.74 (s, 2H), 4.17 (t, J=6.4Hz, 2H), 2.91-2.77 (m, 2H), 2.06 (d, J=1.0 Hz, 3H), 2.03-1.94 (m, 2H).

3-((((3-cyano-4-phenyl-6-(thiophen-2-yl)pyridin-2-yl)thio)methyl)thio)propylacetate. Prepared analogously to4-((((3-cyano-4-phenyl-6-(thiophen-2-yl)pyridin-2-yl)thio)methyl)thio)butylacetate in 26% yield (isolated). ¹H NMR (400 MHz, CHCl₃) δ 7.72 (dd,J=3.8, 1.1 Hz, 1H), 7.66-7.58 (m, 2H), 7.54 (dd, J=4.2, 2.9 Hz, 4H),7.44 (d, J=1.3 Hz, 1H), 7.17 (dd, J=5.0, 3.8 Hz, 1H), 4.55 (s, 2H), 4.18(t, J=6.3 Hz, 2H), 2.84 (t, J=7.3 Hz, 2H), 2.05 (s, 3H), 2.05-1.97 (m,2H). ESI-MS (m/z): 441.0 [M+H]⁺.

3-((((3-cyano-4-phenyl-6-(thiophen-2-yl)pyridin-2-yl)thio)methyl)sulfinyl)propylacetate. Prepared analogously to4-((((3-cyano-4-phenyl-6-(thiophen-2-yl)pyridin-2-yl)thio)methyl)sulfinyl)butylacetate in 90% yield. ¹H NMR (400 MHz, CHCl₃) δ 7.77 (dd, J=3.7, 1.1 Hz,1H), 7.68-7.60 (m, 2H), 7.57 (ddd, J=6.9, 4.5, 2.0 Hz, 4H), 7.51 (s,1H), 7.19 (dd, J=5.0, 3.8 Hz, 1H), 4.81 (d, J=13.1 Hz, 1H), 4.44 (d,J=13.1 Hz, 1H), 4.22 (td, J=6.3, 1.3 Hz, 2H), 3.09 (dt, J=13.0, 8.1 Hz,1H), 2.89 (dt, J=13.1, 7.1 Hz, 1H), 2.28-2.17 (m, 2H), 2.04 (s, 3H).ESI-MS (m/z): 457.1 [M+H]⁺.

SW209273.3-((3-amino-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-2-yl)sulfinyl)propylacetate. Prepared analogously to4-((3-amino-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-2-yl)sulfinyl)butylacetate in 37% yield. ¹H NMR (400 MHz, CHCl₃) δ 7.62-7.52 (m, 5H), 7.45(dd, J=5.0, 1.1 Hz, 2H), 7.41 (s, 1H), 7.10 (dd, J=5.0, 3.7 Hz, 1H),4.65-4.56 (s, 2H), 4.27-4.14 (m, 2H), 3.42-3.25 (m, 1H), 3.15 (dt,J=12.9, 7.7 Hz, 1H), 2.09 (ddd, J=7.5, 6.2, 1.3 Hz, 2H), 2.05 (s, 3H).ESI-MS (m/z): 457.1 [M+H]⁺.

SW209272.3-((3-amino-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-2-yl)sulfonyl)propylacetate. Isolated as the over oxidation product from3-((3-amino-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-2-yl)sulfinyl)propylacetate in 7% yield. ¹H NMR (400 MHz, CHCl₃) δ 7.72 (d, J=3.8 Hz, 1H),7.62-7.53 (m, 3H), 7.54-7.45 (m, 4H), 7.15 (dd, J=5.0, 3.8 Hz, 1H), 5.12(s, 2H), 4.15 (t, J=6.2 Hz, 2H), 3.39-3.19 (m, 2H), 2.25-2.12 (m, 2H),2.03 (s, 3H). ESI-MS (m/z): 457.1 [M+H]⁺.

SW209274.3-((3-amino-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-2-yl)sulfinyl)propan-1-ol.Was prepared analogously to SW209271.4-((3-amino-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-2-yl)sulfinyl)butan-1-olin 84% yield. ¹H NMR (400 MHz, CHCl₃) δ 7.63 (dd, J=3.7, 1.1 Hz, 1H),7.55 (p, J=4.6, 3.2 Hz, 4H), 7.46 (dd, J=5.0, 1.1 Hz, 2H), 7.43 (s, 1H),7.11 (dd, J=5.0, 3.7 Hz, 1H), 4.60 (s, 2H), 3.77 (t, J=5.8 Hz, 2H),3.49-3.33 (m, 1H), 3.21 (dt, J=13.5, 6.9 Hz, 1H), 2.13-1.98 (m, 2H).ESI-MS (m/z): 415.1 [M+H]⁺.

2-((((3-hydroxypropyl)thio)methyl)thio)-4-phenyl-6-(thiophen-2-yl)nicotinonitrile.Prepared analogously to2-((((4-hydroxybutyl)thio)methyl)thio)-4-phenyl-6-(thiophen-2-yl)nicotinonitrilein 98% yield. ¹H NMR (400 MHz, CHCl₃) 7.71 (dd, J=3.8, 1.1 Hz, 1H),7.63-7.57 (m, 2H), 7.55-7.50 (m, 4H), 7.41 (s, 1H), 7.15 (dd, J=5.0, 3.8Hz, 1H), 4.54 (s, 2H), 3.76 (t, J=6.1 Hz, 2H), 2.88 (t, J=7.1 Hz, 2H),1.93 (ddd, J=13.2, 7.1, 6.1 Hz, 2H), 1.88-1.80 (m, 1H). ESI-MS (m/z):399.1 [M+H]⁺.

2-((((3-methoxypropyl)thio)methyl)thio)-4-phenyl-6-(thiophen-2-yl)nicotinonitrile.Sodium hydride (micro spatula tipful) was added to an ice-cooledsolution of2-((((3-hydroxypropyl)thio)methyl)thio)-4-phenyl-6-(thiophen-2-yl)nicotinonitrile(41.6 mg, 0.10 mmol) in DMF (1.0 ml). The mixture was stirred cold for15 minutes before the addition of methyl iodide (34 ml, 0.55 mmol). Themixture was stirred cold in the melting ice-bath for 2 hours, thendiluted with EtOAc and washed several times with water and then brine.The organic layer was dried over Na₂SO₄, filtered and condensed.Purification was carried out on an automated flash chromatography systemin 0-40% EtOAc/hexanes with an isolated yield of 56%. ¹H NMR (400 MHz,CHCl₃) δ 7.72 (dd, J=3.8, 1.1 Hz, 1H), 7.61 (dd, J=6.6, 3.1 Hz, 2H),7.57-7.51 (m, 4H), 7.43 (s, 1H), 7.17 (dd, J=5.0, 3.8 Hz, 1H), 4.55 (s,2H), 3.49 (t, J=6.1 Hz, 2H), 3.34 (s, 3H), 2.85 (t, J=7.3 Hz, 2H), 1.95(ddd, J=13.4, 7.3, 6.1 Hz, 2H). ESI-MS (m/z): 413.1 [M+H]⁺.

2-((((3-methoxypropyl)sulfinyl)methyl)thio)-4-phenyl-6-(thiophen-2-yl)nicotinonitrile.Prepared analogously to4-((((3-cyano-4-phenyl-6-(thiophen-2-yl)pyridin-2-yl)thio)methyl)sulfinyl)butylacetate in 71% yield. ¹H NMR (400 MHz, CHCl₃) δ 7.76 (dd, J=3.8, 1.1 Hz,1H), 7.65-7.58 (m, 2H), 7.56 (td, J=4.6, 2.0 Hz, 4H), 7.49 (s, 1H), 7.18(dd, J=5.0, 3.8 Hz, 1H), 4.73 (d, J=13.1 Hz, 1H), 4.47 (d, J=13.0 Hz,1H), 3.54 (qt, J=9.5, 5.8 Hz, 2H), 3.34 (s, 3H), 3.14 (dt, J=13.1, 7.9Hz, 1H), 2.89 (ddd, J=13.1, 8.0, 6.4 Hz, 1H), 2.20-2.08 (m, 2H). ESI-MS(m/z): 429.1 [M+H]⁺.

SW209276.2-((3-methoxypropyl)sulfinyl)-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-3-amine.Was prepared analogously to SW209329.2-((4-chlorobutyl)sulfinyl)-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-3-amine.Isolated yield=48%. ¹H NMR (400 MHz, CHCl₃) δ 7.64 (ddd, J=7.2, 3.8, 1.7Hz, 2H), 7.58-7.52 (m, 4H), 7.48-7.42 (m, 2H), 7.12 (qd, J=3.7, 1.8 Hz,1H), 4.57 (s, 2H), 3.50 (td, J=6.1, 1.6 Hz, 2H), 3.40-3.29 (m, 4H),3.26-3.13 (m, 1H), 2.09-1.95 (m, 2H). ESI-MS (m/z): 429.1 [M+H]⁺.

3-((((3-cyano-4-phenyl-6-(thiophen-2-yl)pyridin-2-yl)thio)methyl)thio)propylmethanesulfonate. Prepared analogously to4-((((3-cyano-4-phenyl-6-(thiophen-2-yl)pyridin-2-yl)thio)methyl)thio)butylmethanesulfonate in quantitative yield. ¹H NMR (400 MHz, CHCl₃) δ 7.69(t, J=3.3 Hz, 1H), 7.63-7.55 (m, 2H), 7.52 (p, J=3.6, 3.0 Hz, 4H), 7.41(q, J=2.6, 2.2 Hz, 1H), 7.14 (p, J=3.4, 2.5 Hz, 1H), 4.52 (q, J=2.2 Hz,2H), 4.40-4.22 (m, 2H), 2.99 (s, 3H), 2.86 (td, J=7.2, 4.8 Hz, 2H), 2.10(qt, J=6.4, 2.3 Hz, 2H). ESI-MS (m/z): 477.0 [M+H]⁺.

2-((((3-chloropropyl)thio)methyl)thio)-4-phenyl-6-(thiophen-2-yl)nicotinonitrile.Prepared analogously to2-((((4-chlorobutyl)thio)methyl)thio)-4-phenyl-6-(thiophen-2-yl)nicotinonitrile.Purification was performed using an automated flash chromatographysystem in 0-50% EtOAc/hexanes with an isolated yield of 69%. ¹H NMR (400MHz, CHCl₃) δ 7.72 (dd, J=3.7, 1.1 Hz, 1H), 7.64-7.59 (m, 2H), 7.57-7.53(m, 4H), 7.44 (s, 1H), 7.17 (dd, J=5.0, 3.8 Hz, 1H), 4.56 (s, 2H), 3.68(t, J=6.3 Hz, 2H), 2.93 (t, J=7.0 Hz, 2H), 2.15 (p, J=6.7 Hz, 2H).ESI-MS (m/z): 417.0 [M+H]⁺.

2-((((3-chloropropyl)sulfinyl)methyl)thio)-4-phenyl-6-(thiophen-2-yl)nicotinonitrile.Prepared analogously to2-((((4-chlorobutyl)sulfinyl)methyl)thio)-4-phenyl-6-(thiophen-2-yl)nicotinonitrilein 90% yield. ¹H NMR (400 MHz, CHCl₃) δ 7.76 (dd, J=3.8, 1.1 Hz, 1H),7.62 (dq, J=7.1, 2.6, 2.2 Hz, 2H), 7.59-7.53 (m, 4H), 7.51 (s, 1H), 7.18(dd, J=5.0, 3.8 Hz, 1H), 4.74 (d, J=13.1 Hz, 1H), 4.51 (d, J=13.1 Hz,1H), 3.79-3.63 (m, 2H), 3.28-3.16 (m, 1H), 3.04-2.88 (m, 1H), 2.43-2.31(m, 2H). ESI-MS (m/z): 433.0 [M+H]⁺.

SW209330.2-((3-chloropropyl)sulfinyl)-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-3-amine.Prepared analogously to SW209329.2-((4-chlorobutyl)sulfinyl)-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-3-amine.Purification on an automated chromatography system in 0-60%EtOAc/hexanes gave the desired in 88% yield. ¹H NMR (400 MHz, CHCl₃) δ7.57 (h, J=5.7, 5.3 Hz, 1H), 7.45 (t, J=6.0 Hz, 0H), 7.40 (s, 0H), 7.09(t, J=4.4 Hz, 0H), 4.61 (s, 0H), 3.67 (td, J=6.4, 3.2 Hz, 0H), 3.40 (dt,J=14.1, 7.3 Hz, 0H), 3.24 (dt, J=13.1, 7.6 Hz, 0H), 2.25 (p, J=7.0 Hz,0H).

ESI-MS (m/z): 433.0 [M+H]⁺.

2-((((3-fluoropropyl)thio)methyl)thio)-4-phenyl-6-(thiophen-2-yl)nicotinonitrile.Kryptofix 222 (44.4 mg, 0.012 mmol), KF (6.1 mg, 0.10 mmol) and K₂CO₃(3.0 mg, 0.022 mmol) were charged to a vial containing3-((((3-cyano-4-phenyl-6-(thiophen-2-yl)pyridin-2-yl)thio)methyl)thio)propylmethanesulfonate (54.2 mg, 0.11 mmol). DMF (1.1 ml) was added and thereaction was heated at 85° C. for 65 minutes. The cooled mixture wasdiluted with EtOAc and washed several times with water and then brine.The organic layer was dried over Na₂SO₄, filtered and condensed.Yield=96%. Crude product was carried forward. ¹H NMR (400 MHz, CHCl₃) δ7.72 (dd, J=3.7, 1.1 Hz, 1H), 7.64-7.59 (m, 2H), 7.54 (dd, J=4.9, 2.2Hz, 4H), 7.43 (s, 1H), 7.17 (dd, J=5.1, 3.7 Hz, 1H), 4.63 (t, J=5.7 Hz,1H), 4.55 (s, 2H), 4.51 (t, J=5.8 Hz, 1H), 3.67 (t, J=6.3 Hz, 2H), 2.91(dt, J=11.2, 7.1 Hz, 1H), 2.14 (p, J=6.7 Hz, 1H). ESI-MS (m/z): 401.1[M+H]⁺.

2-((((3-fluoropropyl)sulfinyl)methyl)thio)-4-phenyl-6-(thiophen-2-yl)nicotinonitrile.Acetic acid (215 μl) and hydrogen peroxide (16.75 μl, 30% solution inwater) were added to the solution of2-((((3-fluoropropyl)thio)methyl)thio)-4-phenyl-6-(thiophen-2-yl)nicotinonitrile(43.3 mg, 0.034 mmol) in chloroform (215 μl). The reaction mixture wasstirred at 32° C. for 50 min and then diluted with chloroform and waswashed with saturated NaHCO₃ solution and extracted three times withchloroform. The combined organic layers was dried over Na₂SO₄, filteredand concentrated under reduce pressure in 94% yield. ESI-MS (m/z): 417.1[M+H]⁺.

SW209331.2-((3-fluoropropyl)sulfinyl)-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-3-amine.Prepared analogously to SW209329.2-((4-chlorobutyl)sulfinyl)-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-3-amine.The crude mixture was purified preparatively in 4% MeOH/DCM. Isolatedyield=32%. ¹H NMR (400 MHz, CHCl₃) δ 7.68 (dd, J=3.8, 1.1 Hz, 1H), 7.56(q, J=2.7 Hz, 4H), 7.53-7.44 (m, 3H), 7.14 (dd, J=5.1, 3.7 Hz, 1H), 4.65(td, J=5.8, 3.2 Hz, 1H), 4.60 (s, 2H), 4.53 (td, J=5.8, 3.1 Hz, 1H),3.40 (dt, J=13.0, 7.3 Hz, 1H), 3.24 (dt, J=13.1, 7.6 Hz, 1H), 2.19 (dtt,J=26.4, 7.5, 5.7 Hz, 2H). ESI-MS (m/z): 417.1 [M+H]⁺.

2-((((3-cyanopropyl)thio)methyl)thio)-4-phenyl-6-(thiophen-2-yl)nicotinonitrile.A solution of3-((((3-cyano-4-phenyl-6-(thiophen-2-yl)pyridin-2-yl)thio)methyl)thio)propylmethanesulfonate (54.6 mg, 0.11 mmol) and KCN (76.9 mg, 1.18 mmol) inDMF (1.14 ml) was heated at 85° C. for 4 hours. The cooled mixture wasdiluted with EtOAc and washed several times with water and then brine.The organic phase was dried over Na₂SO₄, filtered and condensed.Yield=89%. ¹H NMR (400 MHz, CHCl₃) δ 7.71 (d, J=3.5 Hz, 1H), 7.60 (dt,J=6.4, 2.0 Hz, 3H), 7.54 (qt, J=5.6, 2.5 Hz, 4H), 7.44 (d, J=1.4 Hz,1H), 7.16 (ddd, J=5.2, 3.8, 1.5 Hz, 1H), 4.53 (d, J=1.7 Hz, 2H),2.93-2.82 (m, 2H), 2.52 (td, J=7.1, 1.4 Hz, 2H), 2.09-1.92 (m, 2H).ESI-MS (m/z): 408.1 [M+H]⁺.

2-((((3-cyanopropyl)sulfinyl)methyl)thio)-4-phenyl-6-(thiophen-2-yl)nicotinonitrile.Acetic acid (205 μl) and hydrogen peroxide (15.6 μl, 30% solution inwater) were added to the solution of2-((((3-cyanopropyl)thio)methyl)thio)-4-phenyl-6-(thiophen-2-yl)nicotinonitrile(41.1 mg, 0.10 mmol) in chloroform (205 μl). The reaction mixture wasstirred at 32° C. for 70 min and then diluted with chloroform and waswashed with saturated NaHCO₃ solution and extracted three times withchloroform. The combined organic layers was dried over Na₂SO₄, filteredand concentrated under reduce pressure in 91% yield. ESI-MS (m/z): 424.1[M+H]⁺.

SW209332.4-((3-amino-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-2-yl)sulfinyl)butanenitrile.Prepared analogously to SW209329.2-((4-chlorobutyl)sulfinyl)-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-3-amine.The crude mixture was purified preparatively in 4% MeOH/DCM. Isolatedyield=45%. ¹H NMR (400 MHz, CHCl₃) δ 7.65 (d, J=3.7 Hz, 1H), 7.60-7.51(m, 4H), 7.51-7.44 (m, 3H), 7.13 (t, J=4.4 Hz, 1H), 4.64 (s, 2H), 3.41(dt, J=14.0, 7.3 Hz, 1H), 3.19 (dt, J=13.3, 7.5 Hz, 1H), 2.59 (t, J=7.1Hz, 2H), 2.20 (p, J=7.3 Hz, 2H). ESI-MS (m/z): 424.0 [M+H]⁺.

Example 4 Analysis of the Mechanism of 15-PGDH Inhibition by SW033291and Related Compounds

The following Example provides data relating to the mechanism of actionby which SW033291 inhibits 15-PGDH.

Duplicate titrations of 15-PGDH Inhibitor (SW033291) were run at 4different concentrations of 15-PGDH (24 nM, 12 nM, 6 nM, 3 nM).Reactions contained the indicated concentration of enzyme, 250 μMNAD(+), 25 μM PGE-2, and were assembled and incubated at roomtemperature for 3 minutes.

FIGS. 21 (A-B) show the shift in IC₅₀ value with changing enzymeconcentration. The result is indicative of a tight-binding mode ofinhibition with dependency on enzyme: inhibitor stoichiometry, ratherthan on the absolute concentration of the inhibitor. In all cases, theIC₅₀ values are less than the enzyme concentration, indicating that nMdrug is almost fully bound by the enzyme.

FIGS. 22 (A-B) show the enzymatic activity following dialysis of 15-PGDHand of 15-PGDH treated with SW033291. Graph A) shows activitypre-dialysis and post-dialysis of 15-PGDH protein treated with eitherDMSO or with SW033291. For dialysis, a 500 μL dialysis bag contained 64μg of 15-PGDH at 4 μM concentration, SW033291 at 15 μM concentration,DMSO 1.6%, 1 mM NAD(+), 50 mM Tris pH 7.5, 0.01% Tween 20. The controlreaction omitted SW033291. The 15-PGDH-SW033291 mixture was dialyzedagainst two 1 L buffer exchanges of 12 hours each, with dialysis buffercontaining: 50 mM Tris-HCl pH7.5, 40 mM NaCl, 0.1 mM DTT, 0.01%Tween-20. Both at the start and at the conclusion of dialysis, a 1 μLsample was removed and added to 200 μl of assay buffer of 50 mM Tris-HClpH7.5, 0.01% Tween 20, 150 uM NAD(+), 20 μM PGE2. Generation of NADH wasfollowed in a fluorescent plate reader at 340 nM excitation and 485 nMemission as measured every 30 seconds for 15 minutes. Relative enzymaticactivity of 15-PGDH was determined by the rate of NADH generation overtime [Niesen, 2010 #1676].

Graph B replots the data as % inhibition of 15-PGDH activity in SW033291treated 15-PGDH measured pre-dialysis versus post-dialysis.

Under the conditions of the assay SW033291 inhibited 91% of 15-PGDHpre-dialysis, and 85% of 15-PGDH activity post-dialysis—that is dialysisdid not reverse the inhibition of 15-PGDH. Control 15-PGDH protein thatwas dialyzed in the absence of SW033291 remained fully active.

FIGS. 23 (A-B) show (A) at upper right the reaction rates for 15-PGDH inthe presence of a graded set of increasing concentrations of SW033291.In the graph at upper right P is the NADH concentration as a proxy for15-keto-PGE2. (P+S) is the starting PGE2 concentration of 20 μM. Theassay was carried out in the presence of 10 nM recombinant 15-PGDH. Inthe graph at lower left (B), Vo is the initial velocity of the reactionin the absence of SW033291, and Vi is the initial velocity of thereaction in the presence of the corresponding concentration of SW033291.The line shows the curve generated by fitting the data to the Morrisonequation. The curve fitting yields a calculated a Ki^(App) value of0.1015 nM. The dashed line intersects the X axis at 8.5 nM. Thisrepresents the point at which [inhibitor]=[active enzyme] showing thatthe enzyme preparation contains 85% active enzyme. In the Morrisonequation, Ki is the binding affinity of the inhibitor; [S] is substrateconcentration; and Km is the concentration of substrate at which enzymeactivity is at half maximal. Note that IC₅₀ is the functional strengthof the inhibitor. Whereas the IC₅₀ value for a compound may vary betweenexperiments depending on experimental conditions, the Ki is an absolutevalue. Ki is the inhibition constant for a drug; the concentration ofcompeting ligand in a competition assay which would occupy 50% of thereceptors if no ligand were present.

FIGS. 24 (A-B) show duplicate titrations of 15-PGDH Inhibitor (SW033291)that were run at 6 different concentrations of PGE2 (1.25 uM-40 uM). Inthe graph at top, Y-axis is % inhibition of the reaction by SW033291.The X-axis is the concentration of SW033291 in nM. Reactions contain 5.0nM added 15-PGDH, 250 μM NAD(+), and indicated concentrations of PGE-2,were assembled and incubated at room temperature for 60 minutes. The Kmfor PGE2 is approximately 5 μM, and reactions run with PGE2concentrations below 5 μM go very slowly making it difficult toquantitate inhibition by SW033291. However, in reactions with PGE2 atconcentrations of 5 μM-40 μM, the IC₅₀ for SW033291 is unaffected by theincreasing PGE2 concentration, showing that the inhibition isnoncompetitive.

FIG. 25 shows the structure activity relationships of analogues ofSW033291 versus their IC50 against recombinant 15-PGDH. The opticalisomers of SW033291 were separated by preparative HPLC using a 10 mm×250mm Chiralcel ODH column, 5% isopropanol in hexanes, 1 mL/min. The ‘A’isomer is the faster eluting isomer, and has been assigned as the(−)-(S)-enantiomer. The ‘B’ isomer is the slower eluting isomer and hasbeen assigned as the (+)-(R)-enantiomer.

The analogue family shows that SW033291, with a 4 carbon side chain, is2-fold more potent than SW208080 (5 carbon side chain), 15-fold morepotent than SW208081 (6 carbon side chain), and 100-fold more potentthan SW208079 (1 carbon side chain). SW033291 is also 20-fold morepotent than SW208078, the analogue that converts the sulfoxide group toa sulfone.

FIG. 26 shows structures of additional SW033291 analogs that convert thesulfoxide group to a ketone, an amide, an ester, or a carboxylic acid.Also shown is structure SW206980 that deletes the phenyl ring fromSW033291.

FIGS. 27 (A-C) show graphs that show the level of compound's activity ininducing the 15-PGDH-luciferase fusion reporter in three different testcell line backgrounds, V9m, LS174T, and V503. Each compound was testedat two concentrations, 2.5 μM, and 7.5 μM. Y-axis is luciferaseactivity.

At 2.5 μM-7.5 μM, SW206980, that deletes the phenyl group of SW033291,shows activity comparable to SW033291 in all three reporter lines.

Structures that have converted the sulfoxide group to a ketone, amide,ester, or carboxylic acid show major loss of activity in inducing thereporter.

FIG. 28 shows graphs that show the percent of 15-PGDH enzyme activitythat is inhibited at 2.5 μM and at 7.5 μM by each of the 5 testcompounds. SW206980 that deletes the phenyl group of SW033291, shows atthese concentrations similar potency to SW03291 in inhibiting 15-PGDHactivity.

Structures that have converted the sulfoxide group to a ketone, amide,ester, or carboxylic acid show major loss of activity as 15-PGDHinhibitors.

FIGS. 29 (A-B) show a titration curve that plots percent inhibition of15-PGDH enzyme activity at different concentrations of SW033291 andSW0206980.

FIGS. 30 (A-B) show that SW206980 binds directly to 15-PGDH and markedlyshifts its melting curve. Shown at left is the melt curve of 15-PGDH asreflected by fluorescence of the hydrophobic dye SYPRO Orange. Shown atright is the negative first derivative of the melt curve.

Three conditions are plotted, that of 10 μM 15-PGDH, that of 10 μM15-PGDH plus 10 uM SW206980, and that of 10 μM 15-PGDH plus 125 μM NADHplus 10 μM SW206980. The melting temperature, as reflected by theinflection point of the curve at right is shifted by 20° C., from48-degrees up to 68-degrees, in the presence of SW206980 and NADH,reflecting that SW206980 directly binds to and markedly stabilizes thetertiary structure of 15-PGDH, in a manner requiring the presence of theNADH cofactor.

FIGS. 31 (A-C) show further analogues of SW033291 that build on theprevious finding that removal of the SW033291 phenyl ring (SW206980)retained activity. The new analog (SW206992) adds a nitrogen to theleft-hand ring.

Table 2 provides a comparison of the properties of SW033291, SW206980,and SW206992.

TABLE 2 Summary of three SW033291 analogs SW033291 SW206980 SW206992IC₅₀  1.59 nM  0.97 nM 1.411 nM Time to inhibition (10 nM) ~5 mins ~2mins ~2 mins Δ Tm (NADH) 19° C. 15.5° C. 19° C. Concentration for FullCell ~100 nM >300 nM >1 uM Line Reporter Induction Hepatocyte stabilityStable > couple hrs T1/2 = 80 mins Toxicity >10 μM >7.5 μM >7.5 μM

Time to inhibition refers to the time needed to inhibit the generationof NADH by 15-PGDH from the moment with drug is added into the reactionmix. Delta Tm refers to the shift in melting temperature of recombinant15-PGDH in the presence of drug (with cofactor NADH also present).Concentration of Full Cell Line Reporter Induction refers to theconcentration of drug that needs to be added to reporter cell line toachieve maximal induction of the 15-PGDH-luciferase gene fusion reportercassette, as measured by luciferase assays. Hepatocyte stability refersto the half-life of compound in the presence of hepatocytes in culture.Toxicity refers to the concentration of compound needed to decrease cellnumbers in a cell culture assay.

FIGS. 32 (A-C) show titration of induction by SW033291 of the15-PGDH-luciferase gene fusion reporter in three different cell linebackgrounds. In general between 80-160 nM SW033291 exposure for 24 hoursis needed to induce maximal reporter induction.

FIGS. 33 (A-C) show titration of induction by SW206980 of the15-PGDH-luciferase gene fusion reporter in three different cell linebackgrounds. In general ≥300 nM SW206980 exposure for 24 hours is neededto induce maximal reporter induction.

FIGS. 34 (A-C) show titration of induction by SW206992 of the15-PGDH-luciferase gene fusion reporter in three different cell linebackgrounds. In general ≥1000 nM SW206992 exposure for 24 hours isneeded to induce maximal reporter induction.

FIGS. 35 (A-C) show the shift in the melt curve of recombinant 15-PGDHprotein (10 μM) by 20 uM SW033291, SW206980, and SW206992 in thepresence of the cofactor NAD (+)(100 μM). Control melting temperature is49 degrees centigrade. SW033291 shifts the melting temperature to 63degrees. SW206980 shifts the melting temperature to 61 degrees. SW206992shifts the melting temperature to 59 degrees. Thus all three compoundsdirectly bind to 15-PGDH and markedly increase the melting temperatureof the protein, with the order of the temperature shifts beingSW033291>SW206980>SW206992.

FIGS. 36 (A-B) show the shift in the melt curve of recombinant 15-PGDHprotein (10 uM) by 20 uM SW033291, SW206980, and SW206992 in thepresence of the cofactor NADH (100 uM). Control melting temperature is55 degrees centigrade. SW033291 shifts the melting temperature to 74degrees. SW206980 shifts the melting temperature to 70.5 degrees.SW206992 shifts the melting temperature to 68.5 degrees. Thus all threecompounds directly bind to 15-PGDH and markedly increase the meltingtemperature of the protein, with the order of the temperature shiftsbeing SW033291>SW206980>SW206992.

FIGS. 37 (A-C) show a titration curve of 15-PGDH inhibitor compounds inan assay measuring effect on PGE2 levels in the medium of A549 cellsthat have been stimulated with IL1-beta. Highest PGE2 levels, 3000pg/ml, are achieved with SW033291, with maximal effect attained at 2.5μM compound. Next highest PGE2 level, 2500 pg/ml are achieved withSW206980, with maximal effect attained at 7.5 μM compound. Lowestinduction, to 2100 pg/ml PGE2 is achieved with SW206992, with maximaleffect attained at 2.5 μM. In these reactions, A549 cells weremaintained in F12K medium supplemented with 10% fetal calf serum (FBS)and 50 μg/mL gentamicin in a humidified atmosphere containing 5% CO₂ at37° C. Cells were plated in 24-well plates (0.5 mL per well) at about100,000 cells per well in duplicate and grown for 24 h beforestimulation with IL-1β (1 ng/mL) overnight (16 h) to generate PGE2.SW033291 and its analogs were added at the indicated concentrations, andthe incubation continued for 8 h. Medium was collected, and the level ofPGE2 was analyzed by enzyme immunoassay. Data were analyzed from resultsof three independent experiments.

FIGS. 38 (A-C) show assays of cellular toxicity on A549 cells at 24hours of 15-PGDH inhibitors as assayed by CellTiter-Glo measurement. Noeffect on CellTitre-Glo levels is seen by concentrations of up to 10 μMof SW033291, SW206980, and SW2206992.

FIG. 39 shows structures of 7 SW033291 analogues, SW208064, SW208065,SW208066, SW208067, SW208068, SW208069, SW208070.

Table 3 provides tabular summary of the properties of 4 analogues,SW208064, SW208065, SW208066, SW208067, and in particular lists the IC₅₀for each of these 4 compounds against 2.5 nM recombinant 15-PGDH.

TABLE 3 Summary of four SW033291 analogs from UTSW set 8 SW033291SW2068064 SW208065 SW208066 SW208067 IC₅₀  1.23 nM  151.4 nM   4.865 nM 1.368 nM  2.395 nM Time to ~5 mins inhibition (10 nM) Δ Tm (NADH) 19°C. 5° C. 13° C. 16.5° C. 16.5° C. Concentration ~100 nM ~600 nM  ~100 nM ~100 nM  ~500 nM for Full Cell Line Reporter Induction HepatocyteStable > stability couple hrs Toxicity >10 μM

Time to inhibition refers to the time needed to inhibit the generationof NADH by 15-PGDH from the moment with drug is added into the reactionmix. Delta Tm refers to the shift in melting temperature of recombinant15-PGDH in the presence of drug (with cofactor NADH also present).Concentration of Full Cell Line Reporter Induction refers to theconcentration of drug that needs to be added to reporter cell line toachieve maximal induction of the 15-PGDH-luciferase gene fusion reportercassette, as measured by luciferase assays. Hepatocyte stability refersto the half-life of compound in the presence of hepatocytes in culture.Toxicity refers to the concentration of compound needed to decrease cellnumbers in a cell culture assay.

FIG. 40 provides graphical summary showing the activity of each of thecompounds in inducing a 15-PGDH-luciferase fusion gene reporterintroduced into three different colon cancer cell lines, V9m, LS174T,and V503. Results are measured by assay of luciferase activity afterexposure of cells to compound at either 2.5 μM or 7.5 μM compoundsconcentration.

FIG. 41 provides graphical summary showing the activity of each of thecompounds in inhibiting the enzymatic activity of recombinant 15-PGDHenzyme when compound is added at 2.5 μM and at 7.5 μM. 100% inhibitioncorresponds to complete inhibition of the enzyme.

FIG. 42 shows measurement of IC for inhibiting 2.5 nM of recombinant15-PGDH when incubated across a range of concentrations of SW208064,SW208065, SW208066, and SW208067. Y-axis of each graph records percentinhibition of the 15-PGDH enzymatic activity. 100% Inhibitioncorresponds to complete inhibition of the enzyme. X-axis of each graphrecords the log of the inhibitor concentration expressed in nM.

FIG. 43 shows the dose response curve for induction of a15-PGDH-luciferase fusion gene reporter in the V9m cell line backgroundof SW033291, SW208064, SW208065, SW208066, and SW208067. Concentrationsare in nM.

FIG. 44 Shows titration curves of 15-PGDH inhibitor compounds in anassay measuring effects on PGE2 levels in the medium of A549 cells thathave been stimulated with IL1-beta in the same experimental designdescribed for FIG. 37 . At 100 nM concentration of drug, the highestlevels of PGE2 in the medium are achieved by treating cells withSW208066 or with SW208067, after which the next highest level of PGE2 inthe medium is achieved by treating cells with SW033291.

Example 5 Analysis of Toxicity of SW033291

Table 4 shows a summary of a group of 8-12 week old male FVB mice incontrol or SW033291 treatment arms assessed for toxicity of SW033291,with 6 mice in each arm of the study.

TABLE 4 Baseline Characteristics FVB male mice-8-12 weeks old ToxicityStudy WT-Control WT-Treatment p-value Number 6 6 Sex M M Age (Days)73.7.1 ± 4.7 73.2 ± 5.0 0.465 Weight (gm)   27.5 ± 2.4 26.8 ± 3.1 0.412

FIG. 45 shows the daily weights of a group of 8-12 week old FVB micetreated with vehicle or with SW033291 IP at 5 mg/kg twice daily for 21days. SW033291 was administered in a vehicle of 10% Ethanol, 5%Cremophor EL, 85% D5W at a concentration of 125 ug/200 ul. As shown,both vehicle and drug treated mice show equal weight gain during the 21day period, with no evidence for SW033291 reducing mouse weight. N=6mice in both the SW033291 treated and the vehicle treated arms.

Example 6 Analysis of Effect of SW033291 on Bone Marrow Function

This Example shows effects of SW033291 on bone marrow function.

FIGS. 46 (A-C) show analysis of bone marrow of wild-type mice versusmice that are homozygous genetic knockouts for 15-PGDH (PGDH−/− mice).Total bone marrow cellularity and percent of Sca1+/c-Kit+ cells inlineage negative (SKL) cells are the same in both sets of mice. However,bone marrow from 15-PGDH−/− mice shows an approximately 50% increase innumbers of hematopoietic colonies generated when marrow is plated intomethylcelluose. 15-PGDH knockout mice are denoted by label PGDH mice andby label 15-PGDH. WT denotes wild-type mice.

FIG. 47 shows assay in which bone marrow is harvested from a wild-typemouse, and incubated ex vivo on ice for 2 hours with either SW033291(0.5 μM), or 1 uM PGE2 or 1 μM 16,16-dimethyl PGE2 (dmPGE2). Treatedmarrow is again then plated into methylcellulose for counting ofhematopoietic colonies. SW033291 treated marrow again shows anapproximately 50% increase in the number of bone marrow derived coloniesgenerated. Under these conditions, a lesser increase is seen in marrowtreated with PGE2, and a slightly greater increase is seen in marrowtreated with dmPGE2.

FIGS. 48 (A-C) show a study of C57BL/6J mice treated with IP SW033291administered in a vehicle of 10% Ethanol, 5% Cremophor EL, 85% D5W at adose of 5 mg/kg or 20 mg/kg. Panel A shows mouse bone marrowcellularity, white blood count (wbc), red blood count (rbc) andplatelets counts. Panel B shows percent of Sca1+/c-Kit+ cells in lineagenegative (SKL) cells are unchanged in SW033291 treated mice. Panel Cshows that marrow from SW033291 treated mice gives rise to approximately30% increase in numbers of hematopoietic colonies generated when marrowis plated into methylcelluose. Experimental conditions are noted on thefigure.

FIGS. 49 (A-B) show analysis of marrow from CD45.2 antigen markedC57BL/6J mice that were treated with SW033291 5 mg/kg IP daily for 3doses in a vehicle of 10% Ethanol, 5% Cremophor EL, 85% D5W or that weretreated with vehicle alone. On day 3 mice were sacrificed, marrowflushed and mixed at a 1:1 ratio with vehicle treated CD45.1 marrow. 2million whole BM cells were injected into the tail vein of lethallyirradiated CD45.1 mice and percent chimerism measured via flow cytometryat weeks 8, 12, 16. As shown, at weeks 12 and 16 the percent bloodchimerism of CD45.2 marked cells was significantly increased inrecipient mice whose CD45.2 marked marrow was harvested from SW033291treated donor mice, as opposed to vehicle control treated donor mice. Inother words, marrow from SW033291 treated mice demonstrated long termincreased fitness in competition with control marrow. In particular, atweek 16 CD45.2 harvested from SW033291 treated mice show a significantincrease in contribution to B and T cell populations, suggesting marrowfrom SW033291 treated mice promotes earlier reconstitution of lymphoidpopulations and earlier return to immune competence.

In an additional study, C57BL/6J mice are irradiated with 11Gy on day 0,followed by treatment with SW033291 5 mg/kg IP twice daily (bid) in avehicle of 10% Ethanol, 5% Cremophor EL, 85% D5W, or with vehicle onlyfor 21 days. Mice treated with vehicle or with SW033291 all receive anallograft of marrow from a donor C57BL/6J mouse at a dose of either100,000 cells, 200,000 cells, 500,000 cells. 3 control and 3 SW033291mice are assessed under each condition. The experimental design isdepicted in FIG. 50 .

Table 6 shows the number of surviving mice in each cohort over the first19 days of study. Under the conditions of the mouse colony during thisstudy, control mice receiving 100,000-500,000 cells are all dead betweendays 4-13 of study. In contrast, two SW033291 treated mice receiving500,000 cells remain alive on day 19 of the study and are presumed tohave full hematopoietic reconstitution. Thus treatment with the 15-PGDHinhibitor SW033291 promoted survival of mice receiving a bone marrowtransplant, an observation consistent with SW033291 enabling more rapidand complete hematopoietic reconstitution in the transplanted mice.Other 15-PGDH inhibitors with activity similar to SW033291 would bepredicted to have similar activity in supporting hematopoieticreconstitution. Treatment with SW033291 also enabled mice to besuccessfully transplanted with a smaller inoculum of donor bone marrowthan the 1,000,000 cells that are standardly needed. These observationssuggest SW033291, as well as other similar 15-PGDH inhibitors, is ableto support successful transplantation with smaller numbers of donor stemcells. Such activity would be of particular utility in settings, such astransplantation with umbilical cord stem cells, in which donor cellnumbers are limited. Improved survival of transplanted mice treated withSW033291 suggests efficacy of SW033291, and of similar 15-PGDHinhibitors, as replacements for, or in enabling decreased use of, othertreatments or growth factors commonly employed in support of patientsreceiving bone marrow, hematopoietic stem cell, and cord blood stem celltransplants. Improved survival of transplanted mice treated withSW033291 is consistent with SW033291, and by extension other similar15-PGDH inhibitors, having activity in reducing infections in thetransplanted mice, and/or in promoting recovery of mice intestines fromdamage by radiation, and/or in reducing pulmonary toxicity fromradiation.

TABLE 5 Mouse survival 13- 14- 15- 16- 17- 18- 19- 20- 21- 22- 23- 24-25- 26- 1- Mar. Mar. Mar. Mar. Mar. Mar. Mar. Mar. Mar. Mar. Mar. Mar.Mar. Mar. . . . Apr. Day Day Day Day Day Day Day Day Day Day Day Day DayDay Day Treatment Cell number 0 1 2 3 4 5 6 7 8 9 10 11 12 13 19 Control1 × 10{circumflex over ( )}5 3 3 3 2 0 Control 2 × 10{circumflex over( )}5 3 3 3 3 3 2 1 0 Control 5 × 10{circumflex over ( )}5 3 3 3 3 3 3 21 1 1 3 1 3 0 SW033291 1 × 10{circumflex over ( )}5 3 3 3 3 2 1 0SW033291 2 × 10{circumflex over ( )}5 3 3 3 3 3 3 2 2 2 2 2 2 1 0SW033291 5 × 10{circumflex over ( )}5 3 3 3 3 3 3 3 3 2 2 2 2 2 2 . . .2

Example 7 Analysis of Effect of SW033291 on Radiation Survival

This Example shows studies of the effect of SW033291 in mice receivingwhole body irradiation.

Table 5 shows the results of a study of 15 week old C57BL/6J female miceirradiated with 7Gy, 9Gy, or 11Gy, and receiving daily SW033291 5 mg/kgIP in a vehicle of 10% Ethanol, 5% Cremophor EL, 85% D5W for 7 doses, orreceiving vehicle alone. The table shows the number of mice surviving onsequential days of the study. Under the conditions of the mouse colonyduring this experiment, mice receiving a lethal dose of 11Gy lived 48hours longer if treated with SW033291 than if receiving vehicle control,with control mice all dead on day 8; whereas SW033219 treated mice wereall dead on day 10.

TABLE 6 10/2 10/3 10/4 10/5 10/6 10/7 10/8 10/9 10/10 10/11 10/12 10/1310/23 Radiation Treatment Day Day Day Day Day Day Day Day Day Day DayDay Day Day Dose Arm 0 5 6 7 8 9 10 11 12 13 14 15 16 25  7 Gy Saline 33 3 3 3 3 3 3 3 3 3 3 Looks Healthy SW033291 3 3 3 3 3 3 3 3 3 3 3 3Looks Healthy  9 Gy Saline 3 3 3 3 3 3 3 3 2 1 1 0 SW033291 3 3 3 3 3 33 3 2 2 1 0 11 Gy Saline 3 3 3 2 0 SW033291 3 3 3 3 3 2 0

Table 7 shows the number of mice surviving on sequential days of a studyof mice treated at 11Gy treated with either vehicle control or withSW033291 IP, in a vehicle of 10% Ethanol, 5% Cremophor EL, 85% D5W, withSW033291 administered either at 5 mg/kg daily for 7 days, 5 mg/kg dailythroughout the study, or at 5 mg/kg twice daily for 7 days. Again micetreated with SW033291 on any of these dosing schedules live on average1-2 days longer than mice receiving vehicle control. The activity ofSW033291 in promoting resistance to toxic effects of radiation mayextend to SW033291 and other similar 15-PGDH inhibitors in promotingresistance to other similar toxic insults including but not limited toCytoxan, fludarabine, chemotherapy and immunosuppressive therapy.

TABLE 7 Friday Wed. Thurs. Friday Saturday Sunday Monday Treatment12-Oct 17-Oct 18-Oct 19-Oct 20-Oct 21-Oct 22-Oct Conditions Day 0 Day 5Day 6 Day 7 Day 8 Day 9 Day 10 11 Gy Saline 2 2 2 2 0 (7 days, 1 dosedaily) 11 Gy SW033291 3 3 3 3 2 1 0 (1 dose/daily) for 7 days 11 GySW033291 3 3 3 3 3 2 0 (1 dose/daily, continuous every day) 11 Gy Saline3 3 3 2 1 0 0 (7 days, 2 dose/daily 11 Gy SW033291 3 3 3 3 3 2 0 (7days, 2 dose daily)

Example 8 Analysis of Effect of SW033291 on Liver Regeneration PostPartial Hepatectomy

This Example shows studies assessing the effect of SW033291 on liverregeneration in mice following partial hepatectomy.

FIG. 51 shows a drawing of the anatomy of the mouse liver and of thepartial hepatectomy procedure described in Mitchell et al., NatureProtocols, 3, 1167-1170 (2008), in which the median and left laterallobes are resected, and then liver regeneration is observed viahypertrophy of the remaining right and caudate lobes. The totalresection is of approximately 70% of the mouse liver mass. In thesestudies mice were euthanized using carbon dioxide inhalation. The mousebody was weighed in its entirety. The liver was removed from the mouse;the necrotic remnant from the surgical resection was trimmed; and theregenerated liver was weighed.

FIG. 52 (A-D) show an anatomical view of the mouse liver. Pictures atleft are pre-operative views, and those at right are post-resectionviews. The upper two pictures show anterior view of liver and at leftdisplay the median lobe and a part of the Lateral lobe. The lower twopictures show Inferior view of liver and at left display the Laterallobe. In the partial hepatectomy procedure, the median and lateral lobesare resected as shown at right.

FIG. 53 (A-D) at left reiterates photographs of the immediatepost-hepatectomy views of the mouse liver, with anterior view at top andinferior view at bottom. Figure at top right shows the in situ view ofthe regenerated liver on post-operative day (POD) 10, showinghypertrophy of the remnant right and caudate lobes. Photograph at lowerright shows the anterior view of the regenerated liver after removalfrom the mouse body. Whitish region at the upper right liver edge is thenecrotic stump from the resection, and is trimmed prior to weighing.

The first study was performed in 10 week old male C57BL/6J mice,receiving daily SW033291 5 mg/kg IP in a vehicle of 10% Ethanol, 5%Cremophor EL, 85% D5W, versus vehicle alone, and assessed daily forliver regeneration with 5 control and 5 SW033291 recipient micesacrificed on each time point. In this study, ketamine anesthesia wasemployed.

FIG. 54 (A-B) show micrograph of the hematoxylin and eosin stained liveron post-operative day 3 (POD 3) in the SW033291 treated mouse versus thecontrol mouse, with mitotic figures marked by yellow arrows in theSW033291 treated mouse liver at left and by green arrows in the controlmouse liver at right.

FIG. 55 shows a graph of the number of mitosis per high powered field inlivers of SW033291 treated versus control mice on post-operative daystwo through five (2 D-5 D). Mitotic figures were counted in 10 highpowered fields (40×) from each of 5 livers per SW033291 or control miceper day. SW033291 treated mice demonstrated significantly increasedhepatic mitosis versus controls on days 3 and 4.

Table 8 shows the numbers of mitosis per random high powered fieldcounted in livers of control versus SW033291 treated mice onpost-operative days 2 through 5 (2 D-5 D). SW033291 treated mice showsignificantly increased numbers of mitotic liver cells on post-operativedays 3 and 4.

TABLE 8 Mitotic index (40×) mean + SD Control SW033291 p-value 2D 0.7000± 0.1933 1.240 ± 0.2330 0.0915 3D  2.000 ± 0.4364 6.160 ± 0.3250 <0.00014D  2.560 ± 0.2242 4.560 ± 0.7190 0.0107 5D 0.2000 ± 0.1069 0.2400 ±0.08718 ns

FIG. 56 shows the liver to body weight ratios attained following partialhepatectomy in control versus SW033291 treated C57Bl/6j mice injected at5 mg/kg SW033291 IP daily (qd) starting on post-operative day 0 andcontinuing throughout. Graph displays values from post-operative days2-7 (POD 2-7). The SW033291 qd injection group of mice attain a higherliver to body weight ratio from post-operative days 4-7, with theincrease being statistically significant on post-operative day 4 and day7.

An additional group of mice received SW033291 5 mg/kg twice daily (bid)and were analyzed on post-operative day 3, with the data graphed asPOD3b. This group of mice also showed a statistically significantincrease in liver to body weight ratio compared to control mice.

Another study was performed testing the effects of SW033291 given 5mg/kg IP twice daily (bid) on liver regeneration in C57BL/6J mice. 10mice were used in the control and 10 mice in the drug treated arm foranalysis of each time point of the study. The study again employed 10week old male mice receiving daily SW033291 5 mg/kg IP in a vehicle of10% Ethanol, 5% Cremophor EL, 85% D5W, versus vehicle alone, with 10drug treated and 10 control mice sacrificed daily for comparison. Inthis study ketamine anesthesia was employed.

FIG. 57 shows graph of the liver to body weight ratio attained followingpartial hepatectomy in control versus SW033291 treated C57BL/6J miceinjected at 5 mg/kg SW033291 IP twice daily (bid) starting at 1 hourpost-surgery and continued throughout. Graph displays values frompost-operative days 2-7 (POD 2-7). The SW033291 bid injected group ofmice show a statistically significant higher liver to body weight ratioversus control mice on post-operative days 3, 4, and 7.

FIG. 58 reprises the graph of the liver to body weight ratio attainedfollowing partial hepatectomy in control mice versus in mice treatedwith sw033291 5 mg/kg IP twice daily. In data enclosed within the bluebox, drug was started 1 hour following surgery, and significantincreases in liver to body weight ratio are seen in drug treated micefrom post-operative day (POD) 3 onward. In data enclosed within the redbox, the first dose of sw033291 is delivered commencing 1 hour beforesurgery, and significant increase in liver to body weight ratio is seenas early as post-operative day 1, the day following the surgery.

FIGS. 59 (A-B) show graphs of the serum ALT levels following partialhepatectomy in one control mouse versus one mouse treated with sw033291at 5 mg/kg IP twice daily (bid). Post-operative day 1 values arecompared at left, and post-operative day 2-7 values are compared atright. ALT values are lower in the drug treated mouse.

FIG. 60 shows graph of serum bilirubin levels following partialhepatectomy in one control mouse versus one mouse treated with SW033291at 5 mg/kg IP twice daily (bid) from post-operative days (POD) 1-7.

In another study, SW033291 was tested in the partial hepatectomy modelusing the FVB strain of mice administered SW033291 5 mg/kg IP twicedaily (bid), administered in a vehicle of 10% Ethanol, 5% Cremophor EL,85% D5W, using 5 treated mice versus 5 control mice treated with vehiclealone for analysis at each time point from post-operative day (POD) 1-7.In this study ketamine anesthesia was employed.

FIG. 61 shows a graph of the liver to body weight ratio of attainedfollowing partial hepatectomy in FVB mice treated with 5 mg/kg IPSW033291 versus control mice treated with vehicle alone. SW033291treated mice show increased liver to body weight ratio frompost-operative days 2-7, with the increase being statisticallysignificant on POD, 2,3, 4 and 7.

In another study, SW033291 was tested in a partial hepatectomy modelusing the FVB strain of mice administered SW033291 5 mg/kg IP twicedaily (bid), starting 1 hour before surgery. 10 week old male mice wereemployed, with 10 treated mice and 10 control mice used for analysis ateach time point from post-operative day (POD) 1-7. In this studyisoflurane anesthesia was employed. Vehicle treated 15-PGDH knockout(KO) mice were also used as an additional comparator. FIG. 62 shows agraph depicting the pre-operative body weights of the FVB mice used foranalysis of liver regeneration on post-operative days 2, 3, 4 and 7.SW033291 and control treated mice used on each day are well matched.

FIG. 63 shows a graph depicting the weight of the resected liver segment(resected LWt) from mice treated with either SW033291 or vehicle controland assayed for liver regeneration on post-operative days (POD) 2, 3, 4,and 7. Weight of the resected livers is well matched between control anddrug treated mice on each day, except on day 7, when the weight of theresected liver was greater in the SW033291 treated than in the controlmice.

FIG. 64 shows a graph depicting the liver weights attained(Regenerated_LWt) post partial hepatectomy in SW033291 and control miceon post-operative days 2, 3, 4 and 7 (POD 2, 3, 4, 7). SW033291 treatedmice show significantly greater liver weights versus control mice at alltime points, with SW033291 treated mice having approximately 25% greaterliver weight on post-operative day 7 than control mice.

FIG. 65 shows a graph depicting the liver to body weight ratios attained(LBWR) post partial hepatectomy in SW033291 and control mice onpost-operative days 2, 3, 4 and 7 (POD 2, 3, 4, 7). SW033291 treatedmice show significantly greater liver to body weight ratios versuscontrol mice at all time points, with SW033291 treated mice havingapproximately 20% greater liver to body weight ratio on post-operativeday 7 than control mice.

FIG. 66 shows “box and whisker” plot comparing liver to body weightratio's on post-operative day 4 following partial hepatectomy of FVBmice treated twice daily with SWO33291 5 mg/kg or with vehicle control,with 10 mice in each arm. Thick bars denote population median. Upper boxmargin denotes lower boundary of the highest quartile. Lower box margindenotes upper boundary of the lowest quartile. SW033291 treated miceshow a significantly increased liver to body weight ratio at P=0.004.

FIG. 67 shows “box and whisker” plot comparing liver to body weightratio's on post-operative day 7 following partial hepatectomy of FVBmice treated twice daily with SWO33291 5 mg/kg or with vehicle control,with 10 mice in each arm. Thick bars denote population median. Upper boxmargin denotes lower boundary of the highest quartile. Lower box margindenotes upper boundary of the lowest quartile. SW033291 treated miceshow a significantly increased liver to body weight ratio at P=0.001.

FIG. 68 shows “box and whisker” plot comparing liver to body weightratios on post-operative day 4 following partial hepatectomy of FVB micetreated twice daily with SWO33291 5 mg/kg or with vehicle control, with10 mice in each arm. Also shown is the liver to body weight ratio onpost-operative day 4 of 15-PGDH knockout mice (PGDH-KO) treated withvehicle only. Thick bars denote population median. Upper box margindenotes lower boundary of the highest quartile. Lower box margin denotesupper boundary of the lowest quartile. SW033291 treated mice show asignificantly increased liver to body weight ratio at P=0.001. 15-PGDHknockout mice also show greater a greater liver to body weight ratiothan do vehicle treated 15-PGDH wild-type mice, supporting that theliver regeneration activity of SW033291 is mediated through inhibitionof 15-PGDH. The larger effect of 15-PGDH gene knockout suggests furtherincrease in effect of SW033291 may be attainable with additionalmodification of dosing schedule and delivery.

FIG. 69 shows visualization of S-phase cells following partialhepatectomy on post-operative day 2 in livers of SW033291 treated andvehicle treated control mice. Mice were injected with BrdU at 50 mg/kgIP 2 hours before sacrifice, and then S-phase cells were visualized bystaining the livers with an antibody that detects BrdU that has beenincorporated into DNA. Representative fields at 10× magnification showthe clear increase in numbers of BrdU positive cells in the SW033291treated liver.

FIG. 70 shows high powered (40×) views of representative fields from thestudy of FIG. 69 .

FIG. 71 shows “box and whiskers” plot comparing percent of BrdU positivecells in livers of SW033291 treated versus vehicle control treated miceon post-operative day 2 following partial hepatectomy. Plotted in eachgroup are the percent BrdU positive cells from 100 random high poweredfields (40× magnification) counted as 10 fields from each of 10 drugtreated and each of 10 control vehicle treated mice. Heavy black barsshow median values of each distribution. Upper box margin denotes lowerboundary of the highest quartile. Lower box margin denotes upperboundary of the lowest quartile. SW033291 show a greater than 2-foldincrease in median S-phase cells on post-operative day 2 (P<0.05).

Example 9 Analysis of Effect of SW033291 on Survival FollowingAcetaminophen (Tylenol) Overdose

This Example provides data showing effects of SW033291 in mediatingresistance to lethal doses of the liver toxin acetaminophen (Tylenol).

In the study, 11 week old female C57BL/6J mice are injected IP with asuspension of acetaminophen in phosphate buffered saline administered atthe LD50 dose of 600 mg/kg.

Table 9 provides a tabular summary of the number of mice surviving outof an initial cohort of 6 mice that are all treated with acetaminophen(Tylenol) in phosphate buffered saline administered IP at the LD50 doseof 600 mg/kg.

TABLE 9 Treatment 0 hrs 16 hr 24 hrs 40 hrs 48 hrs 64 hrs 72 hrs 88 hr96 hr 112 hr 120 hr 1 does/daily, First dose immediately after Tylenolinjection Saline 6 6 5 4 3 3 3 3 3 3 3 5 mg/kg 6 6 6 4 4 4 4 4 4 4 4SW033291 2 dose/daily, First dose immediately after Tylenol injectionSaline 6 6 6 4 3 3 3 3 3 3 3 5 mg/kg 6 6 6 4 3 3 3 3 3 3 3 SW033291

Test mice are additionally treated with SW033291 5 mg/kg IP in a vehicleof 10% Ethanol, 5% Cremophor EL, 85% D5W beginning immediately followingacetaminophen and continued once daily, or twice daily. Control mice areadditionally treated with vehicle alone once daily or twice daily.Survival is recorded from the 0 time point of administration ofacetaminophen through 120 hours following. No difference is notedbetween survival of SW033291 treated and control mice.

Table 10 shows a summary of the number of mice surviving out of aninitial cohort of 12 eleven week old C57BL/6J female mice that are alltreated with acetaminophen (Tylenol) in phosphate buffered salineadministered IP at the LD50 dose of 600 mg/kg, Mice are additionallytreated with either SW033291 or vehicle control.

TABLE 10 Survival 0 hrs 16 hr 24 hrs 40 hrs 48 hrs 64 hrs 72 hrs 88 hr96 hr 112 hr 120 hr 5 mg/kg 12 12 12 11 10 10 10 10 10 10 10 SW033291Saline 12 12 12 6 5 5 5 5 5 5 5

SW033291 5 mg/kg was administered IP in a vehicle of 10% Ethanol, 5%Cremophor EL, 85% D5W twice daily (bid) beginning 48 hours prior toacetaminophen injection and continuing for 48 hours followingacetaminophen injection for 9 doses total. At 120 hours postacetaminophen injection, 10 of 12 mice have survived in the SW033291treated cohort versus 5 of 12 mice in the vehicle control treatedcohort, P=0.045 in a one-tailed Fisher's exact test. Thuspre-administration of SW033291 protects from the lethal hepatotoxicityof acetaminophen.

Table 11 shows a summary of the number of mice surviving out of aninitial cohort of 6 eleven week old C57BL/6J female mice that aretreated with acetaminophen (Tylenol) in phosphate buffered salineadministered IP at the LD50 dose of 600 mg/kg.

TABLE 11 Survival 0 hrs 16 hr 24 hrs 40 hrs 48 hrs 64 hrs 72 hrs 88 hr96 hr 112 hr 120 hr 5 mg/kg 6 6 5 4 3 3 3 3 3 3 3 SW033291 Saline 6 6 53 2 2 2 2 2 2 2

Mice are additionally treated with either SW033291 or vehicle control.SW033291 5 mg/kg was administered IP in a vehicle of 10% Ethanol, 5%Cremophor EL, 85% D5W twice daily (bid) beginning 3 hours prior toacetaminophen injection and continuing at time 0 through 48 hoursfollowing acetaminophen injection for 6 total doses. At 120 hours postacetaminophen injection 3 of 6 mice have survived in the SW033291treated cohort versus 2 of 6 mice in the vehicle control treated cohort.

Table 12 shows a summary of the number of mice surviving out of aninitial cohort of 7 C57BL/6J 25 week old female 15-PGDH wild-type (WT)or 7 C57BL/6J 25 week old female 15-PGDH knockout (KO) mice treated withacetaminophen (Tylenol) in phosphate buffered saline administered IP atthe LD50 dose of 600 mg/kg.

TABLE 12 Survival 0 hrs 16 hr 24 hrs 40 hrs 48 hrs 64 hrs 72 hrs 88 hr96 hr 112 hr 120 hr WT 7 7 7 5 4 3 3 3 3 3 3 KO 7 7 7 6 6 6 6 6 6 6 6

At 120 hours post acetaminophen injection, 6 of 7 knockout mice surviveversus 3 of 7 wild-type mice. Increased survival of 15-PGDH knockoutmice is consistent with the survival benefit of SW033291 being mediatedthrough inhibition of 15-PGDH.

Example 10 Analysis of Effect of SW033291 on Dextan Sodium Sulfate (DSS)Induced Colitis

This Example provides data from studies of the effect of SW033291 onprevention of induction of colitis in the dextran sodium sulfate (DSS)treated mouse. In the study, 8-12 week old FVB male mice are fed with 2%DSS in drinking water for days 1-7, and then switched to normal drinkingwater beginning on day 8, and continued through day 22. Mice are treatedwith twice daily SW033291 5 mg/kg IP in a vehicle of 10% Ethanol, 5%Cremophor EL, 85% D5W, at 125 μg/200 ul, versus with vehicle alone.Clinical scoring (body weight, rectal bleeding, stool consistency) isrecorded daily, endoscopic scoring (ulcer number, mucosal thickening,and vascular pattern) is assessed on days 8, 11, 15. Mice are sacrificedon days 1, 8, 15 and 22 for assessment of colon length, colon weight,ulcer number, ulcer area, and crypt damage.

Table 13 shows summary of the baseline properties of age and weight ofthe 24 SW033291 treated mice and the 24 control group mice used in thestudy. Also provided is baseline characteristics of 4 FVB male 15-PGDHknockout (KO) mice that are used as a comparator group.

TABLE 13 FVB PGDH WT/KO male mice 8-12 weeks old DSS Study WT-ControlWT-Treatment KO p-value Number 24 24 4 Sex M M M Age (Days) 74.1 ± 3.7 74.2 ± 4.0  73.9 ± 3.4 0.655 Weight (gm) 26.3 ± 1.19 26.8 ± 1.78 27.4 ±1.4 0.391

FIG. 72 shows a graph of the average changes from baseline weight of thecohort of control versus SW033291 treated mice across the 22 days of thestudy. SW033291 treated mice show greater weight at all time points, andin particular, show faster weight gain after washout of DSS then do thecontrol mice, P=0.001.

FIG. 73 shows a graph of the daily Disease Activity Index (DAI) of thecohort of control versus SW033291 treated mice across the 22 days of thestudy. The Disease Activity Index is calculated as an equally weightedaverage of the change from baseline weight, the consistency of stool,and the presence of rectal bleeding, with each component normalized tospan an identical numerical range. SW033291 treated mice show a lowerDisease Activity Index than do control on each day of the study,P<0.001.

FIG. 74 shows the design of the study in which colonoscopic examinationof the left colon, up to the splenic flexure, was performed on live miceon days 8, 11 and 15, under isoflurane anesthesia. Daily weights ofthese SW033291 treated and untreated mice were also recorded and areshown. In addition, post-mortem colonoscopy of the full colon wasperformed on two SW033291 treated and two control treated mice on day15, with findings confirming that DSS induced ulcerations are largelyconfined to the descending colon distal to the splenic flexure.

FIGS. 75 (A-B) show at bottom left the colon as visualized duringcolonoscopy of a DSS treated control mouse that shows loss of themucosal vascular pattern and a gross ulceration. At bottom right isshown the colonoscopic findings of a DSS treated mouse receivingSW033291, with only a small ulcer and with maintenance of the normalmucosal vascular pattern otherwise. Graph at top shows numbers of ulcerspresent on days 8, 11, and 15 in the control versus SW033291 treatedmice. SW033291 treatment prevents two-thirds of ulcer formation.Additional studies of 15-PGDH knockout mice show that 15-PGDH geneknockout prevents 95% of colon ulcer formation. These findings supportthat the colitis prevention activity of SW033291 is mediated through itsactivity as a 15-PGDH inhibitor, and suggest further modifications ofdrug dosing and delivery may provide added colitis prevention and wouldalso be expected to protect from other forms of intestinal injury thatwould include toxicity from radiation, toxicity from chemotherapy, andchemotherapy induced mucositis.

FIG. 76 shows quantitation of ulcer burden on day 15 of DSS treated miceas determined by embedding the full length of the formalin fixed colonsof mice in paraffin blocks, and then microscopic inspection of a random5 μm section along the full colon length for visualization andmeasurement of ulcerated mucosa. The graph shows that the average lengthof ulcerated mucosa is 4.48 mm per colon section in control mice (N=9mice) and is reduced by 61% to a length of 1.74 mm per colon section inSW033291 (drug) treated mice (N=6 mice), P=0.045. Again, 15-PGDH geneknockout (KO) is highly effective in preventing colon ulceration,supporting that the therapeutic effect of SW033291 is mediated throughinhibition of 15-PGDH.

FIGS. 77 (A-B) show examples of scoring murine colonic mucosa accordingto the Murine Endoscopic Index of Colitis Severity (MEICS) (Becker C. etal. Gut 2005; 54: 950-954). At top (A) is shown the colonoscopicfindings and MEICS scoring for a DSS treated mouse receiving SW033291.At bottom (B) is shown the colonoscopic findings and MEICS scoring of aDSS treated mouse receiving vehicle only.

FIG. 78 shows graphs of the MEICS scores for DSS treated mice receivingSW033291 (treatment) versus vehicle (control). MEICS scores showsignificantly less colitis activity in SW033291 treated mice on days 8,11 and 15 of the study.

In addition to the gross visual inspection and scoring of colitisactivity by the MEICS index, full length colons of mice were formalinfixed and paraffin embedded, and microscopic scoring of crypt damage wasperformed using the 0-4 severity scale of Cooper H S. Et al., LabInvest. 1993; 69:238-249. For this analysis, the colons were dividedinto 3 segments of proximal, middle, and distal colon, eachapproximately 1.6 cm in length, with each segment was further subdividedinto 4 sections each approximately 4 mm in length. For each section thecrypt damage severity score was multiplied by the length in mm of thedamaged area, creating a 0-16 cryptitis severity index. An averagecryptitis severity index was calculated for each segment (proximal,middle, and distal colon), and the summed whole colon cryptitis severityindex was determined on a scale of 0-48 for each mouse colon. Inparallel with the visual MEICS score, the microscopic cryptitis severityindex on day 8 of the DSS protocol was significantly greater in controlmice (value of 9.49) than in the SW033291 treated mice (value of 3.16),P<0.05 (data described but not shown in the figure).

FIG. 79 shows assessment of the effect of SW033291 on maintaining DNAsynthesis in the colonic mucosa of DSS treated mice. Mice were injectedwith BrdU at 100 mg/kg IP 3 hours before sacrifice and then full lengthcolons were formalin fixed and embedded in paraffin. S-phase cells, thathave incorporated BrdU into DNA, were visualized by immuno-fluorescentstaining of 5 μm thick sections with an antibody that detects the BrdU.Colonic crypts were visualized by immuno-fluorescent staining with anantibody to the epithelial marker E-Cadherin. Photographic insets showphotomicrographs of high powered fields taken from the mid-colon on day8 of the DSS protocol from control mice, SW033291 treated mice(treatment) and 15-PGDH knockout mice (KO). Red immune-fluorescenceidentifies BrdU positive nuclei, and green immune-fluorescenceidentifies E-Cadherin positive colonocytes. The number of BrdU positivecell per crypt is determined by counting the number of dual labeled redand green cells per average crypt. Green only cells that are not inS-phase are not counted, and red only cells, that are likely stromalcells outside of crypts, are also not counted. On the photomicrographshown crypts are displayed as vertically oriented in control andSW033291 treated mice, and crypts are displayed as horizontally orientedin the 15-PGDH knockout mice. In the photographs the numbers of S-phasecells are fewest in the control mice and are increased in the SW033291treated mice, and increased further in the knockout mice. In theparticular photographs shown, the crypts from control mice both lackS-phase cells and are also visually decreased in height; whereas, cryptheight is increased in the crypts shown from SW033291 treated mice, andcrypt heights is increased further in the crypts shown from 15-PGDHknockout mice. The graph depicts the sum of the average number of BrdUpositive cells per crypt in the distal colon plus the average number ofBrdU positive cells per crypt middle colons of control (Cn), SW033219treated (Tx), and 15-PGDH knockout mice (KO) on day 1, day 8, and day 15of the DSS treatment protocol. On day 8, SW033291 treated micedemonstrate 5.7-fold greater numbers of BrdU positive cells than docontrol mice, which have lost 85% of the day 1 value of BrdU positivecells per crypt. 15-PGDH knockout mice show no loss of BrdU positivecells in the crypt on day 8, consistent with the protective effect ofSW033291 being mediated by inhibition of 15-PGDH.

Table 14 shows a summary of colon length (in cm) in DSS treated micesacrificed on days 8, 15 and 22, in SW033291 treated mice, versusvehicle treated control mice, versus 15-PGDH knockout (KO) mice, whereshortening of the colon is a measure of disease activity.

TABLE 14 Colon length shortening may be correlated to severity of thecolon ulceration Time Point Control SW033291 KO P-value Baseline 8.3 +0.2 8.4 + 0.2 0.71 Day 8 6.6 + 0.4 6.6 + 0.1 1.0 Day 15 7.1 + 0.1 7.5 +0.1 8.5 + 0.1 0.001 Day 22 7.4 + 0.2 8.6 + 0.3 0.012

Vehicle treated control mice show significantly greater colon shorteningat day 22 versus SW033291 treated mice, P=0.012. This comparison is alsoshown graphically in FIG. 80 .

Table 15 shows a summary on day of sacrifice of mouse weights (gms) andcolon lengths (cm) for DSS treated mice receiving SW033291 or vehiclecontrol.

TABLE 15 Vehicle SW033291 KO Wt @ sacrifice-gm Time Point Baseline26.3 + 0.7 25.9 + 0.7 29.2 + 1.3 Day 8 25.4 + 0.7 26.4 + 0.5 Day 1524.4 + 0.5 25.2 + 0.9 Day 22 * 26.3 + 0.7 28.2 + 0.5 Colon length-cmTime Point Baseline  8.3 + 0.2  8.4 + 0.2  8.5 + 0.1 Day 8  6.6 + 0.4 6.6 + 0.1 Day 15  7.1 + 0.1  7.5 + 0.1 Day 22 *  7.4 + 0.2  8.6 + 0.3

On day 22 SW033291 treated mice show greater body weight and greatercolon lengths, indicative of therapeutic effect of SW033291 inprotecting against DSS induced colitis.

Example 11 Analysis of Effect of SW033291 on Survival of Mice FollowingBone Marrow Transplant

FIG. 81 shows enhanced survival in mice receiving a bone marrowtransplant and also administered the 15-PGDH inhibitor SW033291. FIG.81A shows the design of the study. Mice were irradiated with a bonemarrow ablative dose of 11Gy on day 0, followed by administration ofSW033291 in 5 mg/kg twice daily by intraperitoneal injection in avehicle of 10% Ethanol, 5% Cremophor EL, 85% D5W at a concentration of125 μg/200 μl. A matched control cohort received injections with vehicleonly. On day one mice received an infusion of donor marrow at doses of100,000 cells; 200,000 cells; or 500,000 cells. FIG. 81B shows graphicalsurvival curves for mice transplanted with 100,000 donor cells. FIG. 81Cshows graphical survival curves for mice transplanted with 200,000 donorcells. FIG. 81D shows graphical survival curves for mice transplantedwith 500,000 donor cells. And FIG. 81E shows tabular survival data forall mice in the study on study day 30. Among mice receiving 100,000donor cells, all mice succumb, but SW033291 treated mice show anapproximate doubling of median survival. Among mice receiving 200,000donor cells, all control mice were dead by day 12. In contrast, all micereceiving 200,000 donor cells plus SW033291 survived at 30 days ofobservation and were successfully engrafted. Among mice receiving500,000 donor cells, control mice showed a 37.5% mortality; whereas,mice receiving SW033291 again all survived.

FIG. 82 shows a set of studies conducted on lethally irradiated micethat received 500,000 donor marrow cells and were treated with eitherSW033291 at 5 mg/kg intraperitoneal dose twice daily or with vehiclecontrol, in a design otherwise identical to that used for the studies ofFIG. 81 . FIG. 82A shows measurements on blood and bone marrow on day 5after transplant, with FIG. 82B showing that SW033291 treated mice havesignificantly higher total white count and FIG. 82C showing thatSW033291 treated mice have significantly higher total platelet count.The star symbol denotes P<0.05.

FIG. 83A shows measurements on blood and bone marrow on day 8 aftertransplant. FIG. 83B shows that SW033291 treated mice have significantlyhigher platelet count than control, with drug treated mice having 77,000platelets compared to control mice having 39,500 platelets. The starsymbol denotes P<0.05.

FIG. 84A shows measurements on blood and bone marrow on day 12 aftertransplant. FIG. 84B shows that SW033291 treated mice have significantlyhigher neutrophil counts, with drug treated mice having 332 neutrophilscompared to control mice having 125 neutrophils. FIG. 84C shows that onday 12 after transplant, SW033291 treated mice have significantly higherhemoglobin count than controls, with drug treated mice having hemoglobinlevel of 11.58 and control mice having hemoglobin level of 8.3.Additionally, FIG. 84D shows SW033291 treated mice also havesignificantly greater total white counts than control mice. The starsymbol denotes P<0.05.

FIG. 85A shows measurements on blood and bone marrow on day 18 aftertransplant. SW033291 treated mice have significantly higher total whitecount (FIG. 85B), lymphocyte count (FIG. 85C), and neutrophil count(FIG. 85D), with drug treated mice having 835 neutrophils and controlmice having 365 neutrophils (FIG. 85D). In comparison with counts on day12, administration of SW033291 accelerates recovery of neutrophil countsby nearly 6 days (FIG. 84B versus FIG. 85D). FIG. 84E also shows that onday 18 drug treated mice have significantly higher platelet counts thancontrol mice. Last, day 18, drug treated mice have nearly 4-foldincreased percentage (FIG. 84F) and total numbers (FIG. 85G) of SKLmarked bone marrow stem cells than do control mice, with drug treatedmice having a mean of 4127 SKL marked bone marrow cells compared tocontrol mice having a mean of 967 SKL marked bone marrow cells. The starsymbol denotes P<0.05.

FIG. 86(A) shows measurement of PGE2 (pg of PGE2/mg tissue protein) in 4different mouse tissues (colon, bone marrow, liver, lung) across timefollowing IP injection of SW033291 at 10 mg/kg. Blue bar representsbaseline at time 0, and red bars represent time course of PGE2concentration from 1-12 hours following SW033291 injection. FIG. 86(B)shows time course of PGE2 in control mice injected with vehicle only.

FIG. 87 shows a schema of an experiment in which mice are lethallyirradiated (IR) and 12 hours later receive a transplant (BMT) with CFSEdye labeled bone marrow cells (BM), and the number of transplanted cellsthat home and survive in the bone marrow of the recipient mice are thendetermined by FACS at 16 hours post-transplant. In different arms of theexperiment mice are treated with vehicle, with SW033291 (10 mg/kg IP),or with SW033291 (10 mg/kg IP) plus Indomethacin. Drugs are administeredfollowing radiation, following the transplant, and again at 8 hoursafter the transplant.

FIG. 88 shows a graph illustrating the percent of CFSE dye labeled cellsthat have homed to the bone marrow of mice treated as per the schemadescribed in FIG. 87 . Treating mice with SW033291 concurrent with andfollowing the bone marrow transplant increases numbers of homed cells inthe recipient mouse bone marrow 3-fold. The figure further shows thatthe effect of SW033291 is near completely blocked by indomethacin(Indo+SW0), an inhibitor of COX enzymes the produce prostaglandins. Thisis consistent with the effect of SW033291 being mediated through theinhibition of 15-PGDH and through the resulting increase in tissueprostaglandins.

FIG. 89 shows a schema of an experiment in which mice are lethallyirradiated (IR) and 12 hours later receive a transplant (BMT) with CFSEdye labeled bone marrow cells (BM), and number of transplanted cellsthat home and survive in the bone marrow of the recipient mice are thendetermined by FACS at 16 hours post-transplant. In different arms of theexperiment mice are treated with vehicle, with SW033291 (10 mg/kg IP),with SW033291 (10 mg/kg IP) plus an antagonist of PGE2 receptor EP2(PF-04418948), or with SW033291 (10 mg/kg IP) plus an antagonist of PGE2receptor EP4 (L-161,982). Drugs are administered following radiation,following the transplant, and again at 8 hours after the transplant.

FIG. 90 shows a graph illustrating the percent of CFSE dye labeled cellsthat have homed to the bone marrow of mice treated as per the schemadescribed in FIG. 89 . Treating mice with SW033291 concurrent with andfollowing the bone marrow transplant increases numbers of homed cells inthe recipient mouse bone marrow 2-fold. The figure further shows thatthe effect of SW033291 is near completely blocked by an antagonist tothe EP4 receptor (EP4+SW0), and is partially blocked by an antagonist ofthe EP2 receptor (EP2+SW0). This is consistent with the effect ofSW033291 being mediated through the inhibition of 15-PGDH and throughthe resulting increase of tissue prostaglandins, including PGE2.

FIG. 91 shows a schema of an experiment in which mice are injected withSW033291 twice daily IP at 10 mg/kg for 5 doses. 2 hours following thelast dose bone marrow is harvested and sorted into SKL marked cells thatare hematopoietic stem cell enriched and into CD45(−) cells that arebone marrow stroma cells. RNA was extracted and gene expressiondetermined relative to levels in mice injected with vehicle control.

FIGS. 92 (A-B) show graph illustrating induction of gene expression inbone marrow SKL cells and bone marrow stromal cells of SW033291 treatedmice. Mice administered SW033291 show a 3 fold induction in RNAexpression of CXCL12 and SCF in bone marrow SKL cells, and show agreater than 4-fold induction of CXCL12 and SCF in CD45(−) bone marrowstromal cells.

FIG. 93 illustrates a schema of an experiment in which immune deficientNSG mice are lethally irradiated (IR) and 12 hours later receive atransplant with CFSE dye labeled buffy coat cells from human umbilicalcord blood (UCB), and number of transplanted cells that home and survivein the bone marrow of the recipient mice are then determined by FACS at16 hours post-transplant. In different arms of the experiment mice aretreated with vehicle or with SW033291. Drugs are administered followingradiation, following the transplant, and again at 8 hours after thetransplant.

FIG. 94 illustrates a graph showing the percent of CFSE dye labeledhuman umbilical cord buffy coat cells that have homed to the bone marrowof mice treated as per the schema described in FIG. 94 . Treating micewith SW033291 at the time of and following the transplant with buffycoat from human umbilical cord blood (UCB) increases numbers of homedhuman cells in the recipient mouse bone marrow nearly 2-fold.

Example 12 Analysis of Activity of Isomers of SW033291, a 15-PGDHInhibitor

FIG. 95 illustrates isomers of SW033291. Shown at top is that Sulfoxides(the S═O group) are stereogenic such that SW033291 exists as a racemicmixture of two non-interconverting isomers. These enantiomers wereseparated by preparative HPLC to provide isomers designated as isomer A(first eluting) and isomer B (second eluting). A 1 cm Chiralcel-ODHcolumn resolves the enantiomers using isopropanol in hexanes. Arepresentative analytical HPLC trace is shown at bottom. Isomer A wasfound to consist predominantly of the levorotatory enantiomer: [a]D=−97(c=0.22, acetone). Isomer B was found to consist predominantly of thedextrorotatory enantiomer: [a]D=+95 (c=0.22, acetone). X-raycrystallography has assigned (−) isomer A as the S isomer and B as the(R) isomer (as shown in Table 1)

FIG. 96 illustrates plots showing thermal denaturation of recombinant15-PGDH protein using Differential Scanning Fluorimetry with SYPROOrange. Top panel graphs Relative Fluorescence Units (RFU) versustemperature for recombinant 15-PGDH in presence of DMSO control,SW033291, SW033291-A (isomer A), or SW033291-B (isomer B). Lower panelgraphs -d(RFU)/dT. The melting temperatures are indicated in the tableat lower left. Only isomer B shows that activity of SW033291 in bindingto 15-PGDH and shifting the melting temperature from 50.5° C. to 68° C.

FIGS. 97 (A-C) illustrate inhibition of recombinant 15-PGDH proteinenzymatic activity by SW033291 stereogenic isomers A and B. Graphed atleft is the percent inhibition of enzyme activity of recombinant 15-PGDHprotein versus concentration of SW033291, and the IC₅₀ for inhibition.Graphed at right is the percent inhibition of recombinant 15-PGDH versusconcentration of SW033291 isomer A (top) of isomer B (bottom). As shownisomer B has 40% lower IC₅₀ than the parent racemic mixture; whereas;isomer A shows an IC₅₀ 36 fold higher than the racemic mixture. Theseresults are consistent with the activity of SW033291 in inhibiting15-PGDH largely due to the activity of isomer B. The small residualactivity of isomer A may be due to possible 3% contamination with someresidual isomer B.

FIG. 98 is a graph showing the activity in inducing activity of a15-PGH-luciferase fusion protein reporter in the Vaco-9M (V9M) cell linebackground of SW033291 isomers A and B. Full activity in this assay isseen for SW033291 isomer B at 0.1 μM added compound. Some residualactivity is seen for isomer B when added at 3.2 μM concentration. Thesmall residual activity of isomer A may be due to possible 3%contamination with some residual isomer B.

Example 13

Analysis of in Silico Docking of SW033291 onto 15-PGDH

FIG. 99 illustrates images showing docked pose of SW209415 onto the15-PGDH crystal structure. The model shows that the sulfoxide moiety onSW209415 coordinates with Tyr-151 in 15-PGDH, that is required forcatalytic activity of 15-PGDH and that is strictly conserved across thefamily of short chain dehydrogenases. Moreover, the SW209415 sulfoxideis also coordinated with Ser-138, that is also required for catalyticactivity of 15-PGDH and that is highly conserved across the family ofshort chained dehydrogenases. Thus, it is expected that the corestructure of inhibitor would broadly interact and inhibit any shortchain dehydrogenase whose catalytic function depended on a tyrosineanalogous to 15-PGDH Tyr-151 and/or a serine analogous to Ser-138.

Example 14 Analysis of Analogues of Lead Compounds SW033291, a 15-PGDHInhibitor

This Example provides data on a group of structural analogues ofSW033291. Data provided includes level of induction of a15-PGDH-luciferase fusion gene reporter, recorded as % increasedluciferase activity over basal level, in three colon cancer cell lines,V9m, V503, and LS174T, engineered to contain the reporter, and treatedthe compound (i.e., Values are recorded on a scale where 100 indicatesof doubling of luciferase activity over baseline level).

FIG. 100 shows chemical structures of a set of seven compounds,designated SW209123 through SW209129, that are structurally related toSW033291.

FIG. 101 shows the activity of each compound SW209123 through SW209129in inhibiting the enzymatic activity of recombinant 15-PGDH protein inin vitro assays, with the percent inhibition graphed on the Y-axis andthe Log of the compound concentration in nM graphed on the X-axis. Theinhibition curve for SW033291 is shown as a comparator. The IC₅₀ foreach compound is recorded. Two compounds, SW209124 and SW209125, showlower IC₅₀, and hence higher activity, than does SW033291.

FIG. 102 shows the activity of compounds SW209123 through SW209129 ininducing PGE2 as assayed in the medium of A549 cells that have firstbeen activated with IL-1beta. For comparison, also shown is induction ofPGE2 as assayed in the medium of A549 cells that have first beenactivated with IL-1beta and then treated with SW033291. Graphed are theeffects of exposing the IL-1beta stimulated A549 cells to increasingcompound concentrations of 4 nM, 20 nM, 100 nM, 500 nM, and 2500 nM. Forcomparison PGE2 levels are shown for DMSO treated A549 cells and forA549 cells treated with IL-1beta only. In this assay, SW209124 andSW209125 show increased activity relative to SW033291 in this assay.

FIG. 103 shows activity of compound SW209123 through SW209129 ininducing luciferase activity in a reporter cell line constructed fromthe Vaco 9M (V9M) colon cancer cell line into which a renilla luciferasecassette has been targeted into the last coding exon of the endogenous15-PGDH gene to create an in frame fusion gene encoding a15-PGDH-luciferase fusion protein. Activity of SW033291 in the assay isalso shown. Graphed are results of treating the reporter cells with DMSOor with compounds at concentrations of 39.0635 nM, 78.124 nM, 156.25 nM,312.5 nM, 625 nM, 1250 nM, and 2500 nM.

FIG. 104 shows activity of compound SW209123 through SW209129 ininducing luciferase activity in a reporter cell line constructed fromthe LS174T colon cancer cell line into which a renilla luciferasecassette has been targeted into the last coding exon of the endogenous15-PGDH gene to create an in frame fusion gene encoding a15-PGDH-luciferase fusion protein. Activity of SW033291 in the assay isalso shown. Graphed are results of treating the reporter cells with DMSOor with compounds at concentrations of 39.0635 nM, 78.124 nM, 156.25 nM,312.5 nM, 625 nM, 1250 nM, and 2500 nM.

FIG. 105 shows activity of compound SW209123 through SW209129 ininducing luciferase activity in a reporter cell line constructed fromthe Vaco 503 (V503) colon cancer cell line into which a renillaluciferase cassette has been targeted into the last coding exon of theendogenous 15-PGDH gene to create an in frame fusion gene encoding a15-PGDH-luciferase fusion protein. Activity of SW033291 in the assay isalso shown. Graphed are results of treating the reporter cells with DMSOor with compounds at concentrations of 39.0635 nM, 78.124 nM, 156.25 nM,312.5 nM, 625 nM, 1250 nM, and 2500 nM.

FIG. 106 shows the activity of each compound SW209123 through SW209129in inhibiting the enzymatic activity of recombinant 15-PGDH protein inin vitro assays, with the percent inhibition graphed on the Y-axis andcompound is added at either 2.5 μM or 7.5 μM. Activity of SW033291 inthe assay is also shown.

Example 15

FIG. 107 shows chemical structures of a set of thirteen compounds,designated set 20 with individual compound identifiers ranging fromSW209271 through SW209283, that are structurally related to SW033291.For each compound the molecular weight, tPSA, and C Log P is also shown.

FIGS. 108 and 109 shows the activity of each compound SW209271 throughSW209283 in inhibiting the enzymatic activity of recombinant 15-PGDHprotein in in vitro assays, with the percent inhibition graphed on theY-axis and the Log of the compound concentration in nM graphed on theX-axis. The IC₅₀ for each compound is recorded.

FIG. 110 shows the activity of compounds SW209271 through SW209283 ininducing PGE2 as assayed in the medium of A549 cells that have firstbeen activated with IL-1beta. For comparison, also shown is induction ofPGE2 as assayed in the medium of A549 cells that have first beenactivated with IL-1beta and then treated with SW033291. Graphed are theeffects of exposing the IL-1beta stimulated A549 cells to increasingcompound concentrations of 4 nM, 20 nM, 100 nM, 500 nM, and 2500 nM. Forcomparison PGE2 levels are shown for DMSO treated A549 cells and forA549 cells treated with IL-1beta only.

FIG. 111 shows activity of compounds SW209271 through SW209283 ininducing luciferase activity in a reporter cell line constructed fromthe Vaco 9M (V9M) colon cancer cell line into which a renilla luciferasecassette has been targeted into the last coding exon of the endogenous15-PGDH gene to create an in frame fusion gene encoding a15-PGDH-luciferase fusion protein. Activity of SW033291 in the assay isalso shown. Graphed are results of treating the reporter cells with DMSOor with compounds at concentrations of 19.5 nM, 39.0635 nM, 78.124 nM,156.25 nM, 312.5 nM, 625 nM, and 1250 nM.

FIG. 112 shows activity of compounds SW209271 through SW209283 ininducing luciferase activity in a reporter cell line constructed fromthe LS174T colon cancer cell line into which a renilla luciferasecassette has been targeted into the last coding exon of the endogenous15-PGDH gene to create an in frame fusion gene encoding a15-PGDH-luciferase fusion protein. Activity of SW033291 in the assay isalso shown. Graphed are results of treating the reporter cells with DMSOor with compounds at concentrations of 19.5 nM, 39.0635 nM, 78.124 nM,156.25 nM, 312.5 nM, 625 nM, and 1250 nM.

FIG. 113 shows activity of compound SW209271 through SW209283 ininducing luciferase activity in a reporter cell line constructed fromthe Vaco 503 (V503) colon cancer cell line into which a renillaluciferase cassette has been targeted into the last coding exon of theendogenous 15-PGDH gene to create an in frame fusion gene encoding a15-PGDH-luciferase fusion protein. Activity of SW033291 in the assay isalso shown. Graphed are results of treating the reporter cells with DMSOor with compounds at concentrations of 19.5 nM, 39.0635 nM, 78.124 nM,156.25 nM, 312.5 nM, 625 nM, and 1250 nM.

FIG. 114 shows chemical structures of two previously describedcompounds, SW209125 and SW208436, along with a set of five compounds,designated set 21 with individual compound identifiers ranging fromSW209239 through SW209333, that are structurally related to SW033291.For each compound the molecular weight, tPSA, and C Log P is also shown.

FIG. 115 shows the activity of compounds SW209125, SW208436 and set 21compounds ranging from SW209239 through SW209333 in inhibiting theenzymatic activity of recombinant 15-PGDH protein in in vitro assays,with the percent inhibition graphed on the Y-axis and the Log of thecompound concentration in nM graphed on the X-axis. The IC50 for eachcompound is recorded.

FIG. 116 shows chemical structures of a set of six compounds, designatedset 23, with individual compound identifiers ranging from SW209415through SW209420, that are structurally related to SW033291. For eachcompound the molecular weight, tPSA, and C Log P is also shown.

FIG. 117 shows the activity of set 23 compounds ranging from SW209415through SW209420 in inhibiting the enzymatic activity of recombinant15-PGDH protein in in vitro assays, with the percent inhibition graphedon the Y-axis and the Log of the compound concentration in nM graphed onthe X-axis. The IC₅₀ for each compound is recorded.

FIG. 118 shows the activity of selected set 21 compounds ranging fromSW209239 through SW2093332 and set 23 compounds ranging from SW209415through SW209420 in inducing PGE2 as assayed in the medium of A549 cellsthat have first been activated with IL-1beta. For comparison, also shownis induction of PGE2 as assayed in the medium of A549 cells that havefirst been activated with IL-1beta and then treated with SW033291.Graphed are the effects of exposing the IL-1beta stimulated A549 cellsto increasing compound concentrations of 4 nM, 20 nM, 100 nM, 500 nM,and 2500 nM. For comparison PGE2 levels are shown for DMSO treated A549cells and for A549 cells treated with IL-1beta only.

FIG. 119 shows activity of SW209125, SW208436 and set 21 compoundsranging from SW209239 through SW209333 in inducing luciferase activityin a reporter cell line constructed from the Vaco 9M (V9M) colon cancercell line into which a renilla luciferase cassette has been targetedinto the last coding exon of the endogenous 15-PGDH gene to create an inframe fusion gene encoding a 15-PGDH-luciferase fusion protein. Activityof SW033291 in the assay is also shown. Graphed are results of treatingthe reporter cells with DMSO or with compounds at concentrations of 19.5nM, 39.0635 nM, 78.124 nM, 156.25 nM, 312.5 nM, 625 nM, and 1250 nM.

FIG. 120 shows activity of SW209125, SW208436 and set 21 compoundsranging from SW209239 through SW209333 in inducing luciferase activityin a reporter cell line constructed from the LS174T colon cancer cellline into which a renilla luciferase cassette has been targeted into thelast coding exon of the endogenous 15-PGDH gene to create an in framefusion gene encoding a 15-PGDH-luciferase fusion protein. Activity ofSW033291 in the assay is also shown. Graphed are results of treating thereporter cells with DMSO or with compounds at concentrations of 19.5 nM,39.0635 nM, 78.124 nM, 156.25 nM, 312.5 nM, 625 nM, and 1250 nM.

FIG. 121 shows activity of SW209125, SW208436 and set 21 compoundsranging from SW209239 through SW209333 in inducing luciferase activityin a reporter cell line constructed from the Vaco 503 (V503) coloncancer cell line into which a renilla luciferase cassette has beentargeted into the last coding exon of the endogenous 15-PGDH gene tocreate an in frame fusion gene encoding a 15-PGDH-luciferase fusionprotein. Activity of SW033291 in the assay is also shown. Graphed areresults of treating the reporter cells with DMSO or with compounds atconcentrations of 19.5 nM, 39.0635 nM, 78.124 nM, 156.25 nM, 312.5 nM,625 nM, and 1250 nM.

FIG. 122 shows activity of set 23 compounds ranging from SW209415through SW209420 in inducing luciferase activity in a reporter cell lineconstructed from the Vaco 9M (V9M) colon cancer cell line into which arenilla luciferase cassette has been targeted into the last coding exonof the endogenous 15-PGDH gene to create an in frame fusion geneencoding a 15-PGDH-luciferase fusion protein. Activity of SW033291 inthe assay is also shown. Graphed are results of treating the reportercells with DMSO or with compounds at concentrations of 39.0625 nM,78.125 nM, 156.25 nM, 312.5 nM, 625 nM, 1250 nM, and 2500 nM.

FIG. 123 shows activity of set 23 compounds ranging from SW209415through SW209420 in inducing luciferase activity in a reporter cell lineconstructed from the LS174T colon cancer cell line into which a renillaluciferase cassette has been targeted into the last coding exon of theendogenous 15-PGDH gene to create an in frame fusion gene encoding a15-PGDH-luciferase fusion protein. Activity of SW033291 in the assay isalso shown. Graphed are results of treating the reporter cells with DMSOor with compounds at concentrations of 39.0625 nM, 78.125 nM, 156.25 nM,312.5 nM, 625 nM, 1250 nM, and 2500 nM.

FIG. 124 shows activity of set 23 compounds ranging from SW209415through SW209420 in inducing luciferase activity in a reporter cell lineconstructed from the Vaco 503 (V503) colon cancer cell line into which arenilla luciferase cassette has been targeted into the last coding exonof the endogenous 15-PGDH gene to create an in frame fusion geneencoding a 15-PGDH-luciferase fusion protein. Activity of SW033291 inthe assay is also shown. Graphed are results of treating the reportercells with DMSO or with compounds at concentrations of 39.0625 nM,78.125 nM, 156.25 nM, 312.5 nM, 625 nM, 1250 nM, and 2500 nM.

FIG. 125 reprises the structures of SW209125, set 20 compound SW209279,and set 23 compounds SW209415 and SW209418.

FIG. 126 shows repeat testing of the activity of SW209125, set 20compound SW209279, and set 23 compounds SW209415 and SW209418, ininducing PGE2 as assayed in the medium of A549 cells that have firstbeen activated with IL-1beta. For comparison, also shown is induction ofPGE2 as assayed in the medium of A549 cells that have first beenactivated with IL-1beta and then treated with SW033291. Graphed are theeffects of exposing the IL-1beta stimulated A549 cells to increasingcompound concentrations of 4 nM, 20 nM, 100 nM, 500 nM, and 2500 nM. Forcomparison PGE2 levels are shown for DMSO treated A549 cells and forA549 cells treated with IL-1beta only.

FIG. 127 shows testing of the activity of SW209125, set 20 compoundSW209279, and set 23 compounds SW209415 and SW209418, in inducing PGE2as assayed in the medium of DLD1 cells in media supplemented witharachidonic acid. For comparison, also shown is induction of PGE2 asassayed in the medium of DLD1 cells in media supplemented witharachidonic acid and treated with SW033291. Graphed are the effects ofexposing the DLD1 cells in media with arachidonic acid to increasingcompound concentrations of 4 nM, 20 nM, 100 nM, 500 nM, and 2500 nM. Forcomparison PGE2 levels are shown for DMSO treated DLD1 cells and forDLD1 cells treated with arachidonic acid (AA) only.

FIG. 128 shows activity of SW033291, SW209125, set 20 compound SW209279,and set 23 compounds SW209415 and SW209418, in inducing luciferaseactivity in a reporter cell line constructed from the Vaco 9M (V9M)colon cancer cell line into which a renilla luciferase cassette has beentargeted into the last coding exon of the endogenous 15-PGDH gene tocreate an in frame fusion gene encoding a 15-PGDH-luciferase fusionprotein. Activity of SW033291 in the assay is also shown. Graphed areresults of treating the reporter cells with DMSO or with compounds atconcentrations ranging from (right to left) of 2.4 nM up to 2500 nM.

FIG. 129 shows activity of SW033291, SW209125, set 20 compound SW209279,and set 23 compounds SW209415 and SW209418, in inducing luciferaseactivity in a reporter cell line constructed from the LS174T coloncancer cell line into which a renilla luciferase cassette has beentargeted into the last coding exon of the endogenous 15-PGDH gene tocreate an in frame fusion gene encoding a 15-PGDH-luciferase fusionprotein. Activity of SW033291 in the assay is also shown. Graphed areresults of treating the reporter cells with DMSO or with compounds atconcentrations ranging from (right to left) of 2.4 nM up to 2500 nM.

FIG. 130 shows activity of SW033291, SW209125, set 20 compound SW209279,and set 23 compounds SW209415 and SW209418 in inducing luciferaseactivity in a reporter cell line constructed from the Vaco 503 (V503)colon cancer cell line into which a renilla luciferase cassette has beentargeted into the last coding exon of the endogenous 15-PGDH gene tocreate an in frame fusion gene encoding a 15-PGDH-luciferase fusionprotein. Activity of SW033291 in the assay is also shown. Graphed areresults of treating the reporter cells with DMSO or with compounds atconcentrations ranging from (right to left) of 2.4 nM up to 2500 nM.

FIG. 131 shows chemical structures of a set of seven compounds,designated set 24, with individual compound identifiers ranging fromSW209427 up to SW209513, that are structurally related to SW033291. Foreach compound the molecular weight, tPSA, and C Log P is also shown.Also shown is the repeated structure of SW209415.

FIG. 132 shows the activity of each compound in set 24 with compoundnumbers from SW209427 up to SW209513 in inhibiting the enzymaticactivity of recombinant 15-PGDH protein in in vitro assays, with thepercent inhibition graphed on the Y-axis and the Log of the compoundconcentration in nM graphed on the X-axis. The IC₅₀ for each compound isrecorded. Also shown is the repeated assay of SW209415.

FIG. 133 shows activity of set 24 compounds, with compound numbers fromSW209427 up to SW209513, in inducing luciferase activity in a reportercell line constructed from the Vaco 9M (V9M) colon cancer cell line intowhich a renilla luciferase cassette has been targeted into the lastcoding exon of the endogenous 15-PGDH gene to create an in frame fusiongene encoding a 15-PGDH-luciferase fusion protein. Activity of SW033291in the assay is also shown. Graphed are results of treating the reportercells with DMSO or with compounds at concentrations of 19.5 nM, 39.0635nM, 78.125 nM, 156.25 nM, 312.5 nM, 625 nM, 1250 nM, and 2500 nM.

FIG. 134 shows activity of set 24 compounds, with compound numbers fromSW209427 up to SW209513, in inducing luciferase activity in a reportercell line constructed from the LS174T colon cancer cell line into whicha renilla luciferase cassette has been targeted into the last codingexon of the endogenous 15-PGDH gene to create an in frame fusion geneencoding a 15-PGDH-luciferase fusion protein. Activity of SW033291 inthe assay is also shown. Graphed are results of treating the reportercells with DMSO or with compounds at concentrations of 19.5 nM, 39.0635nM, 78.125 nM, 156.25 nM, 312.5 nM, 625 nM, 1250 nM, and 2500 nM.

FIG. 135 shows dose response curves for inhibition of enzymatic activityof recombinant 15-PGDH (Y-axis) versus log of the nM concentration ofracemic SW209415 (top graph), or of the (−) isomer of SW209415 (bottomleft graph), or of the (+) isomer of SW209415 (bottom right graph). IC₅₀for racemic SW209415 is 2.6 nM. IC₅₀ for (+) SW209415 is 1.3 nM. IC₅₀for (−) SW209415 is 164 nM.

FIG. 136 shows that the enantiomers of SW209415 can be separatedchromatographically using a semi-preparative Chiralpak AD HPLC columnwith methanol as the mobile phase at 2.5 ml/min.

FIG. 137 illustrates a graph illustrating the induction of PGE2 that issecreted into cell culture media of A549 cells that are treated with:DMSO alone; IL1-beta alone; IL1-beta plus racemic SW209415 (labeledSW209415); IL1-beta plus (−) SW209415 (labeled SW209415 (−)); IL1-betaplus (+) SW209415 (labeled SW209415 (+)); or with IL1-beta plus SW033291(labeled SW033291). Each of the 15-PGDH inhibitor drugs was tested asconcentrations of 4 nM, 20 nM, 100 nM, 500 nM, and 2500 nM.

FIGS. 138 (A-D) illustrate graphs showing induction of PGE2 (pg ofPGE2/mg tissue protein) in 4 different mouse tissues (colon, bonemarrow, liver, lung) across time following IP injection of SW209415 at10 mg/kg. Blue bar represents baseline at time 0, and red bars representtime course from 1-12 hours following SW209415 injection.

FIG. 139 shows schema of an experiment in which female C57BL/6J mice arelethally irradiated (IR) and 12 hours later receive a transplant withCFSE dye labeled bone marrow cells from a donor C57BL/6J female mouse,and the number of transplanted cells that home and survive in the bonemarrow of the recipient mice are then determined by FACS at 16 hourspost-transplant. In different arms of the experiment mice are treatedwith vehicle, or with SW033291 10 mg/kg IP, or with SW209415 (racemate)at 10 mg/kg IP. Drugs are administered following radiation, followingthe transplant, and again at 8 hours after the transplant.

FIG. 140 illustrates a graph showing the percent of CFSE dye labeleddonor bone marrow cells that have homed to the bone marrow of recipientmice treated as per the schema described in FIG. 139 . Treating micewith SW033291 (labeled 291) or with SW209415 (labeled 415) at the timeof and following the transplant increases numbers of homed donor cellsin the recipient mouse bone marrow by approximately 2.5-fold.

FIG. 141 shows activity of (+) SW209415 in increasing PGE2 in mousetissues. Shown is measurement by Elisa of PGE2 (ng PGE2/mg of tissueprotein) from 4 mouse tissues: lung, liver, colon and bone marrow. Micewere injected IP with either vehicle control (blue bars), or (+)SW209415 at 0.5 mgkg (yellow bars), 1.5 mg/kg (green bars), or 5 mg/kg(orange bars). Tissues were harvested for PGE2 assay at either 2 hour (2h) or 3 hour (3 h) time points following drug injection. (+) SW209415shows induction of PGE2 in all 4 tissues tested, with activity seen at 2hours, and peak at 3 hours, and with (+) SW209415 induced peak PGE2levels approximately double those of vehicle control.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims. All patents, publications andreferences cited in the foregoing specification are herein incorporatedby reference in their entirety.

1. A compound having the formula (V):

wherein n=0-2; R₁ and R₃ are the same or different and are each selectedfrom the group consisting of:

R₂ is N or CR₇; R₄ is selected from the group consisting of H, Cl, F,NH₂, and N(R₆)₂; R₅ is selected from the group consisting of branched orlinear alkyl including —(CH₂)n₁CH₃ (n₁=0-7),

wherein n₂=0-6 and X is any of the following: CF_(y)H_(z) (y+z=3),CCl_(y)H_(z) (y+z=3), OH, OAc, OMe, R₆, OR₆, CN, N(R₆)₂,

(n₃=0-5, m=1-5), and

(n₄=0-5); each R₆ and R₇ are the same or different and are one or moresubstituent selected from the group consisting of hydrogen, substitutedor unsubstituted C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, C₃-C₂₀aryl, heterocycloalkenyl containing from 5-6 ring atoms, (wherein from1-3 of the ring atoms is independently selected from N, NH, N(C₁-C₆alkyl), NC(O)(C₁-C₆ alkyl), O, and S), heteroaryl or heterocyclylcontaining from 5-14 ring atoms, (wherein from 1-6 of the ring atoms isindependently selected from N, NH, N(C₁-C₃ alkyl), O, and S), C₆-C₂₄alkaryl, C₆-C₂₄ aralkyl, halo, silyl, hydroxyl, sulfhydryl, C₁-C₂₄alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀ aryloxy, acyl(including C₂-C₂₄ alkylcarbonyl (—CO-alkyl) and C₆-C₂₀ arylcarbonyl(—CO-aryl)), acyloxy (—O-acyl), C₂-C₂₄ alkoxycarbonyl (—(CO)—O-alkyl),C₆-C₂₀ aryloxycarbonyl (—(CO)—O-aryl), C₂-C₂₄ alkylcarbonato(—O—(CO)—O-alkyl), C₆-C₂₀ arylcarbonato (—O—(CO)—O-aryl), carboxy(—COOH), carboxylato (—COO⁻), carbamoyl (—(CO)—NH₂), C₁-C₂₄alkyl-carbamoyl (—(CO)—NH(C₁-C₂₄ alkyl)), arylcarbamoyl (—(CO)—NH-aryl),thiocarbamoyl (—(CS)—NH₂), carbamido (—NH—(CO)—NH₂), cyano (—CN),isocyano (—N⁺C⁻), cyanato (—O—CN), isocyanato (—O—N⁺═C⁻), isothiocyanato(—S—CN), azido (—N═N⁺═N⁻), formyl (—(CO)—H), thioformyl (—(CS)—H), amino(—NH₂), C₁-C₂₄ alkyl amino, C₅-C₂₀ aryl amino, C₂-C₂₄ alkylamido(—NH—(CO)-alkyl), C₆-C₂₀ arylamido (—NH—(CO)-aryl), sulfanamido (—SO2NR2where R is independently H, alkyl, aryl or heteroaryl), imino (—CR═NHwhere R is hydrogen, C₁-C₂₄ alkyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄aralkyl, etc.), alkylimino (—CR═N(alkyl), where R=hydrogen, alkyl, aryl,alkaryl, aralkyl, etc.), arylimino (—CR═N(aryl), where R=hydrogen,alkyl, aryl, alkaryl, etc.), nitro (—NO₂), nitroso (—NO), sulfo(—SO₂—OH), sulfonato (—SO₂—O⁻), C₁-C₂₄ alkylsulfanyl (—S-alkyl; alsotermed “alkylthio”), arylsulfanyl (—S-aryl; also termed “arylthio”),C₁-C₂₄ alkylsulfinyl (—(SO)-alkyl), C₅-C₂₀ arylsulfinyl (—(SO)-aryl),C₁-C₂₄ alkylsulfonyl (—SO₂-alkyl), C₅-C₂₀ arylsulfonyl (—SO₂-aryl),sulfonamide (—SO₂—NH2, —SO₂NY₂ (wherein Y is independently H, aryl oralkyl), phosphono (—P(O)(OH)₂), phosphonato (—P(O)(O⁻)₂), phosphinato(—P(O)(O⁻)), phospho (—PO₂), phosphino (—PH₂), polyalkyl ethers(—[(CH₂)_(n)O]_(m)), phosphates, phosphate esters [—OP(O)(OR)₂ whereR=H, methyl or other alkyl], groups incorporating amino acids or othermoieties expected to bear positive or negative charge at physiologicalpH, and combinations thereof; R₃ is not hydrogen if R₁ is H, anunsubstituted thiophene, or an unsubstituted thiazole and R₅ is butyl;or R₃ is not an unsubstituted phenyl if R₁ is H, or an unsubstitutedphenyl, thiophene, or thiazole and R₅ is benzyl or (CH₂)n₅(CH₃)(n₅=0-5); or a pharmaceutically acceptable salt thereof.
 2. The compoundof claim 1, wherein R₂ is N or CH.
 3. The compound of of claim 1,wherein R₁ is a substituted or unsubstituted heterocyclyl containing 5-6ring atoms.
 4. The compound of of claim 1, wherein R₁ is a substitutedor unsubstituted thiophene, thiazole, oxazole, imidazole, pyridine, orphenyl.
 5. The compound of of claim 1, wherein n is
 1. 6. The compoundof of claim 1, wherein R₁ is a substituted or unsubstituted thiophene,thiazole, oxazole, imidazole, pyridine, or phenyl.
 7. The compound ofclaim 1, wherein R₃ is selected from the group consisting of H,substituted or unsubstituted aryl, a substituted or unsubstitutedcycloalkyl, and a substituted or unsubstituted heterocyclyl, alkyl, orcarboxy including carboxylic acid (—CO2H), carboxy ester (—CO₂alkyl) andcarboxamide [—CON(H)(alkyl) or —CO₂N(alkyl)₂].
 8. The compound of claim1, including a compound including formula (V₁)

wherein n=0-2; R₃ is selected from the group consisting of:

R₂ is N or CR₇; R₄ is selected from the group consisting of H, Cl, F,NH₂, and N(R₆)₂; R₅ is selected from the group consisting of branched orlinear alkyl including —(CH₂)n₁CH₃ (n₁=0-7),

wherein n₂=0-6 and X is any of the following: CF_(y)H_(z) (y+z=3),CCl_(y)H_(z) (y+z=3), OH, OAc, OMe, R₆, OR₆, CN, N(R₆)₂,

(n₃=0-5, m=1-5), and

(n₄=0-5); each R₆ and R₇ are the same or different and are one or moresubstituent selected from the group consisting of hydrogen, substitutedor unsubstituted C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, C₃-C₂₀aryl, heterocycloalkenyl containing from 5-6 ring atoms, (wherein from1-3 of the ring atoms is independently selected from N, NH, N(C₁-C₆alkyl), NC(O)(C₁-C₆ alkyl), O, and S), heteroaryl containing from 5-14ring atoms, (wherein from 1-6 of the ring atoms is independentlyselected from N, NH, N(C₁-C₃ alkyl), O, and S), C₆-C₂₄ alkaryl, C₆-C₂₄aralkyl, halo, silyl, hydroxyl, sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀ aryloxy, acyl (including C₂-C₂₄alkylcarbonyl (—CO-alkyl) and C₆-C₂₀ arylcarbonyl (—CO-aryl)), acyloxy(—O-acyl), C₂-C₂₄ alkoxycarbonyl (—(CO)—O-alkyl), C₆-C₂₀ aryloxycarbonyl(—(CO)—O-aryl), C₂-C₂₄ alkylcarbonato (—O—(CO)—O-alkyl), C₆-C₂₀arylcarbonato (—O—(CO)—O-aryl), carboxy (—COOH), carboxylato (—COO⁻),carbamoyl (—(CO)—NH₂), C₁-C₂₄ alkyl-carbamoyl (—(CO)—NH(C₁-C₂₄ alkyl)),arylcarbamoyl (—(CO)—NH-aryl), thiocarbamoyl (—(CS)—NH₂), carbamido(—NH—(CO)—NH₂), cyano (—CN), isocyano (—N⁺C⁻), cyanato (—O—CN),isocyanato (—O—N⁺═C⁻), isothiocyanato (—S—CN), azido (—N═N⁺═N⁻), formyl(—(CO)—H), thioformyl (—(CS)—H), amino (—NH₂), C₁-C₂₄ alkyl amino,C₅-C₂₀ aryl amino, C₂-C₂₄ alkylamido (—NH—(CO)-alkyl), C₆-C₂₀ arylamido(—NH—(CO)-aryl), sulfanamido (—SO2NR2 where R is independently H, alkyl,aryl or heteroaryl), imino (—CR═NH where R is hydrogen, C₁-C₂₄ alkyl,C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, etc.), alkylimino(—CR═N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, aralkyl, etc.),arylimino (—CR═N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.),nitro (—NO₂), nitroso (—NO), sulfo (—SO₂—OH), sulfonato (—SO₂—O⁻),C₁-C₂₄ alkylsulfanyl (—S-alkyl; also termed “alkylthio”), arylsulfanyl(—S-aryl; also termed “arylthio”), C₁-C₂₄ alkylsulfinyl (—(SO)-alkyl),C₅-C₂₀ arylsulfinyl (—(SO)-aryl), C₁-C₂₄ alkylsulfonyl (—SO₂-alkyl),C₅-C₂₀ arylsulfonyl (—SO₂-aryl), sulfonamide (—SO₂—NH2, —SO₂NY₂ (whereinY is independently H, aryl or alkyl), phosphono (—P(O)(OH)₂),phosphonato (—P(O)(O⁻)₂), phosphinato (—P(O)(O⁻)), phospho (—PO₂),phosphino (—PH₂), polyalkyl ethers (—[(CH₂)_(n)O]_(m)), phosphates,phosphate esters [—OP(O)(OR)₂ where R=H, methyl or other alkyl], groupsincorporating amino acids or other moieties expected to bear positive ornegative charge at physiological pH, and combinations thereof; R₃ is nothydrogen if R₅ is butyl; or R₃ is not an unsubstituted phenyl if R₅ isbenzyl or (CH₂)n₅(CH₃) (n₅=0-5); or a pharmaceutically acceptable saltsthereof.
 9. The compound of claim 8, wherein R₂ is N or CH.
 10. Thecompound of claim 8, wherein n is
 1. 11. The compound of claim 8,wherein R₃ is selected from the group consisting of H, substituted orunsubstituted aryl, a substituted or unsubstituted cycloalkyl, and asubstituted or unsubstituted heterocyclyl, alkyl, or carboxy includingcarboxylic acid (—CO2H), carboxy ester (—CO₂alkyl) and carboxamide[—CON(H)(alkyl) or —CO₂N(alkyl)₂].
 12. The compound of claim 1, having aformula selected from the group consisting of:

or pharmaceutically acceptable salts thereof.
 13. The compound of claim1, having the formula:

or a pharmaceutically acceptable salt thereof.
 14. The compound of claim1, having the formula:

or a pharmaceutically acceptable salt thereof.
 15. The compound of claim1, having the formula:

or a pharmaceutically acceptable salts thereof.
 16. The compound ofclaim 1, consisting essentially of the (+) optical isomer of a compoundof the formula:

or a pharmaceutically acceptable salts thereof. 17-79. (canceled)
 80. Amethod of treating a disease, disorder, or condition where it is desiredto modulate 15-PGDH activity, the method comprising: administering thecompound of claim 1 to a subject in need of treatment.